Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach

AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE substrates have been identified by immunoprecipitation and mass spectrometry approaches, and the full range of targets is yet to be determined in mammalian cells. We describe here the first example of global chemoproteomic screening and substrate validation for HYPE-mediated AMPylation in mammalian cell lysate. Through quantitative mass-spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification, we identified a total of 25 AMPylated proteins, including the previously validated substrate endoplasmic reticulum (ER) chaperone BiP (HSPA5), and also novel substrates involved in pathways of gene expression, ATP biosynthesis, and maintenance of the cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.Covalent posttranslational modification (PTM) of hydroxyl-containing amino acids in proteins by adenosine monophosphate (AMP), called AMPylation or adenylylation, was first discovered almost a half century ago as a mechanism controlling the activity of bacterial glutamine synthetase (1). This unusual PTM was unknown in eukaryotes until it was identified in 2009 in the context of bacterial infection, when Yarbrough et al. reported AMPylation of host small GTPases by bacterial virulence factor Vibrio outer protein S (VopS) from Vibrio parahemeolyticus. In this context, AMPylation precludes interactions with downstream binding partners and causes actin cytoskeleton collapse leading to cell death (2). Since then, the field of AMPylation has grown substantially, with reports describing AMPylation activity of other bacterial effectors, like Immunoglobulin binding protein A (IbpA) in Histophilus somni (3) and Defects in Rab1 recruitment protein A (DrrA) in Legionella pneumophila (4). These new bacterial AMPylators share a common substrate class (small GTPases); however, they differed in the identity of their catalytic residues and architecture of their active sites. Accordingly, bacterial AMP transferases have been classified as either filamentation induced by cyclic AMP (FIC) or adenylyl transferase (AT)¹ domain containing enzymes, with catalytic His or Asp residues, respectively.Although adenylylation has been most extensively described in the context of bacterial infection, there is a growing interest in elucidating the scope of this PTM in a native eukaryotic context. Among the ca. 3000 FIC proteins identified so far by sequence alignment, only a single enzyme has been identified in eukaryotes: Huntingtin-associated protein E (HYPE), also known as FICD. HYPE is conserved from C. elegans to humans, and mRNA expression data suggest that it is present at low levels in all human tissues (3). Apart from the catalytic FIC domain, the protein consists of one transmembrane helix and two tetratricopeptide repeat motifs that point to localization at a membrane and amenability toward protein–protein interactions, respectively. We recently added to this picture by solving the first crystal structure of Homo sapiens HYPE (5), illustrating that the only human FIC is substantially different from its bacterial cousins (6, 7). HYPE was shown to form stable asymmetric dimers supported by the extended network of contacts exclusive to the FIC domains, while the tetratricopeptide repeat motifs have a more flexible arrangement and appear to be exposed for protein–protein interactions in the vicinity of the membrane. In addition, we confirmed the similarity of the active site architecture to other FIC proteins for which a crystal structure is available, with the catalytic loop comprising the invariant catalytic His³⁶³ (8), and further substantiated the role of a critical residue Glu²³⁴ in an inhibitory helix (9) that may be responsible for regulating HYPE enzymatic activity.Various catalytic activities have been demonstrated for FIC proteins, including nucleotide (AMP, GMP, and UMP) transfer as well as phosphorylation and phosphocholination (10–13). We and others (3, 5, 14, 15) have demonstrated that HYPE can function in protein AMPylation, although the activity of the wild-type (WT) enzyme is very weak, consistent with active site obstruction by Glu²³⁴. It is hypothesized that this intramolecular inhibition can be relieved by specific but as yet unknown protein–protein interactions or by the removal of the conserved Glu. Indeed, the E234G mutation substantially boosts HYPE''s activity as demonstrated by the elevated auto-AMPylation of HYPE itself (5, 9) and a few of its recently reported substrates, including the ER chaperone BiP in vivo (14, 15) and several histone proteins in vitro (16, 17). HYPE activity was initially implicated in visual neurotransmission in flies (18) and later in regulation of the unfolded protein response (UPR) in transfected cells, although there is limited consensus over the mechanism (14, 15). Most recently, it has been proposed that HYPE activity might have a role in regulation of gene expression; however, the mechanistic details remain to be elucidated (17).AMPylation profiling is not a trivial task (19), and several strategies have emerged over the past few years ranging from labeling with radioactive ATP (2, 3) and immunoprecipitation with AMPylation-specific antibodies (20, 21) to mass spectrometry (MS) approaches focused on AMP fragmentation (22, 23). Although these methods contributed significantly to developments in the field, they also suffer from certain drawbacks, including low sensitivity, high background, limited quantitative power, and limited amenability to high-throughput (HT) substrate identification. In contrast, chemoproteomic strategies involving application of substrate analogues (substrate probes) equipped with small and inert chemical handles in combination with sensitive detection by MS can facilitate rapid visualization and/or robust enrichment of modified proteins and can provide superior performance in HT profiling of numerous challenging PTMs (24). AMPylation-specific substrate probes have been developed, and their robust performance was evaluated in vitro, albeit to date only in the context of bacterial effector-mediated AMPylation (25–27). We previously showed that a bioorthogonal substrate probe (26) is well tolerated in the active site of human HYPE and, moreover, that it has potential for chemoproteomic profiling of HYPE substrates in vitro when combined with ligation through copper-catalyzed azide alkyne cycloaddition (CuAAC) to a dedicated capture reagent decorated with a biotin affinity handle and carboxytetramethylrhodamine (TAMRA) fluorophore (5).Herein, we present the first global AMPylation profile in a native eukaryotic context utilizing a bioorthogonal ATP analogue and chemoproteomic methodology. We first demonstrate efficient enrichment and fast visualization of potential HYPE substrates in cell lysates by in-gel fluorescence, followed by robust identification via shotgun proteomics on a QExactive mass spectrometer. Furthermore, we extensively validate candidate substrates via HYPE titration and ATP competition experiments with a quantitative MS-based readout, as well as Western blotting and direct MS/MS evidence for AMP modification. Finally, we analyze HYPE interaction partners in vivo, providing a link between our discoveries in lysates and a physiologically relevant context, delivering the first experimentally validated library of HYPE substrate proteins.