Michael F. Albers

Covalent Protein Labeling by Enzymatic Phosphocholination.

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.

Efficient Synthesis and Applications of Peptides containing Adenylylated Tyrosine Residues

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.

Inter-kingdom signaling by the Legionella Quorum Sensing Molecule LAI-1 modulates cell migration through an IQGAP1-Cdc42-ARHGEF9-dependent pathway

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9.

Exploring adenylylation and phosphocholination as post-translational modifications.

Editing the translations: Adenylylation and phosphocholination have recently been found as important post-translational modifications used by pathogenic bacteria during the infection process. This review discusses the combined use of chemical handles and specific antibodies for the identification of previously unknown substrates of these post-translational modifications in infected host cells.

Adenylylation, MS, and proteomics--Introducing a "new" modification to bottom-up proteomics.

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.

Amino Acid Building Blocks for Fmoc Solid-Phase Synthesis of Peptides Phosphocholinated at Serine, Threonine, and Tyrosine

Phosphocholination of eukaryotic host cell proteins has recently been identified as a novel post-translational modification important for bacterial pathogenesis. Here, we describe the first straightforward synthetic strategy for peptides containing phosphocholinated serine, threonine, or tyrosine residues using preformed functional amino acid building blocks, fully compatible with standard Fmoc solid-phase peptide synthesis.

Amino Acid Building Blocks for Efficient Fmoc Solid-Phase Synthesis of Peptides Adenylylated at Serine or Threonine

The first straightforward building block based (non-interassembly) synthesis of peptides containing adenylylated serine and threonine residues is described. Key features include final global acidolytic protective group removal as well as full compatibility with standard Fmoc solid-phase peptide synthesis (SPPS). The described Thr-AMP SPPS-building block has been employed in the synthesis of the Thr-adenylylated sequence of human GTPase CDC42 (Ac-SEYVP-T(AMP)-VFDNYGC-NH(2)). Further, we demonstrate proof-of-concept for the synthesis of an Ser-adenylylated peptide (Ac-GSGA-S(AMP)-AGSGC-NH(2)) from the corresponding adenylylated serine building block.

Enzymatic phosphocholination as a tool for protein labeling

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 ...Dynamic modeling showed that the topology of fatty-acid betaoxidation makes this pathway intrinsically vulnerable to substrate overload: at a high influx of palmitoyl-CoA into the pathway the flux dropped and intermediate CoA-esters accumulated extremely(Van Eunen et al., 2013 PLoS Comput Biol). We show here that inborn errors in fatty-acid metabolism aggravate the risk of amitochondrial catastrophe.We applied the previously constructed dynamic model to study the impact of multiple acyl-CoA dehydrogenase deficiency(MADD) and medium-chain acyl-CoA dehydrogenase deficiency(MCADD) on the kinetics of fatty acid oxidation. We explored the relation between the deficiencies and metabolite profiles and calculated which profiles might enhance the risk of pathway overload. MADD patients show accumulation of acylcarnitines acrossall chain lengths. In contrast, MCADD patients accumulate the medium-chain acylcarnitines. A linear non-competition model could not explain this, as it predicted exclusive accumulation of longer chain-length metabolites in MADD. This provides the first experimental evidence that molecular competition at the enzyme level is physiologically relevant for fatty-acid oxidation. Subsequently,this more realistic competition model was fitted to either mouse liver data or to disease-specific patient plasma data. When the substrate concentration was varied, both MADD and MCADD enhanced the accumulation of intermediate metabolite sand the flux declined already at lower substrate concentrations compared to the model without enzyme deficiencies.We hypothesize that the pathway structure of the beta-oxidationin which substrates compete for enzymes, is at the basis of the disease phenotypes associated with enzyme deficiencies.