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A New Chemical Handle for Protein AMPylation at the Host–Pathogen Interface

Post-translational modification (PTM) of proteins plays a role in the regulation and control of all major cellular processes. This phenomenon involves the (generally enzymatic) covalent attachment of a specific moiety, such as a phosphate, carbohydrate, or lipid, to a particular amino acid side chain of a target protein. This in turn alters the protein’s properties, for example enzymatic activity, subcellular localization, proteolytic stability, or ability to interact with other proteins. A common characteristic of PTMs is their dynamic nature, and the existence of enzymatic activities that reverse PTMs can allow regulation of cellular signaling. A number of recent reports have demonstrated how chemical approaches can be particularly powerful in unraveling the process of PTM, with protein lipidation providing a paradigm example. 3] AMPylation, the covalent attachment of adenosine monophosphate (AMP) to the hydroxyl side chains of proteins through a phosphodiester bond, is yet another class of PTM. It was initially observed in the 1970s in the context of metabolic regulation of Escherichia coli glutamine synthetase by glutamine synthetase adenylyl transferase, but its potential beyond metabolic regulation went largely unexplored for the next four decades. It is only very recently that the field of protein AMPylation has been revitalized by the discovery that pathogenic bacteria utilize specific effector proteins (AMPylators) to catalyze the transfer of AMP from adenosine triphosphate (ATP) to host guanosine triphosphate hydrolases (GTPases) and disrupt their interactions with cognate binding partners. Host GTPases are a major target of pathogenic bacteria as they are involved in various defense mechanisms, including apoptosis, pyroptosis, phagocytosis, and phagosome trafficking. Key bacterial AMPylators characterized to date include VopS, a cytotoxin of Vibrio parahaemolyticus, that AMPylates Rho family GTPases at a conserved threonine residue; IbpA secreted by Histophilus somni, which AMPylates the same target protein but modifies a tyrosine residue instead of a threonine; and finally DrrA (also known as SidM), a Legionella pneumophila effector that AMPylates Rab1 GTPases, also at a tyrosine. Both VopS and IbpA mediate AMPylation by using a so-called Fido catalytic motif (canonical sequence: HPFx[D/ E]GN[G/K]R) and target the host-cell cytoskeleton, whereas DrrA utilizes an adenylyl transferase domain (canonical sequence: Gx11DxD) [11] and targets host-cell protein trafficking. In 2011, the first de-AMPylating enzyme, or “protein AMPylase”, was discovered in L. pneumophila, thus confirming the importance and the dynamic nature of post-translational AMPylation. 14] Apart from its role in pathogenicity, there is growing evidence that AMPylation might operate as a general intracellular signaling mechanism in normal cell function. Bacterial Fido domain proteins are involved in cell division, whereas HYPE, a human Fido motif protein, might regulate the interactions of small GTPases with their binding partners. Furthermore, metabolic labeling experiments with P-a-ATP show numerous radioactive protein bands in human cell lysates; this suggests the presence of additional AMPylating enzymes. AMPylation is rapidly emerging as a fundamental mechanism that serves to regulate protein–protein interactions and cell signaling in normal cells and during the invasion of mammalian cells by bacteria. However, without robust tools to identify and manipulate both AMP transferases and their AMPylated protein substrates, our understanding of this complex signaling network will remain superficial. So far, the majority of reported and predicted AMPylators in bacteria and eukaryotic cells have been found through bioinformatic methods by utilizing sequence/structure similarities to Fido and adenylyl transferase domains. In turn, AMPylated substrate proteins have been identified by the combination of radioactive-isotope labeling and targeted mass spectrometry analysis both on the MS and MS/MS level. 16] Two additional developments that complement the above-mentioned approaches are the elucidation of the mass spectrometric fragmentation characteristics of AMPylation (based on the analysis of synthetic peptides modified at serine, threonine, and tyrosine residues) and the generation of a polyclonal antibody specific for threonine AMPylation. In a very recent report, a potentially more general, chemistry-based methodology for labeling AMP transferase substrates was proposed by Hang, Orth and co-workers. Specifically, the authors took advantage of the chemical proteomic approach that has previously been applied to mapping the substrates of other PTMs, such as glycosylation, prenylation, and acylation. The process relies on the enzymatic transfer of a chemical handle, a so-called bioorthogonal reporter (usually an azide or an alkyne), from a synthetic substrate analogue onto the target protein. This biotransformation is followed by a nonenzymatic but highly chemoselective reaction, in this case a Cu-catalyzed azide–alkyne cycloaddition (CuAAC), with an appropriate ligation partner in order to introduce secondary labels (affinity tag or fluorophore) that allows target enrichment and/or detection. It is important to emphasize that this [a] Dr. M. Broncel, Dr. R. A. Serwa, Dr. E. W. Tate Department of Chemistry, Imperial College London South Kensington Campus Exhibition Road, London SW7 2AZ (UK) E-mail : e.tate@imperial.ac.uk