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

Malgorzata Broncel,Remigiusz A Serwa,Tom D Bunney,Matilda Katan,Edward W Tate

doi:10.1074/mcp.o115.054429

Abstract

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.

Highlights

From the ‡Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK; §Division of Biosciences, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK; ¶Current address: The Institute of Cancer Research, Division of Cancer Biology, 237 Fulham Road, London SW3 6JB, UK
We previously showed that a bioorthogonal substrate probe [26] is well tolerated in the active site of human HYPE and, that it has potential for chemoproteomic profiling of HYPE substrates in vitro when combined with ligation through coppercatalyzed azide alkyne cycloaddition (CuAAC) to a dedicated capture reagent decorated with a biotin affinity handle and carboxytetramethylrhodamine (TAMRA) fluorophore [5]
We previously demonstrated that HYPE activity can be efficiently visualized utilizing a chemoenzymatic strategy, with Yn-6-ATP and a capture reagent, Az-TB [5]

Summary

Introduction

Phate (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. 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 Rab recruitment protein A (DrrA) in Legionella pneumophila [4] These new bacterial AMPylators share a common substrate class (small GTPases); they differed in the identity of their catalytic residues and architecture of their active sites. It has been proposed that HYPE activity might have a role in regulation of gene expression; the mechanistic details remain to be elucidated [17]

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Results

Conclusion