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

Broncel, Malgorzata; Serwa, Remigiusz A.; Tate, Edward W.

doi:10.1002/cbic.201100743

Cited by 17 publications

(11 citation statements)

References 28 publications

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“…The distribution of these candidate substrates' molecular weights agreed with previous findings using gel-based AMPylation assays (Fig. 2E, 2F) (2,3,12,14). The annotations showed that many of the VopS and IbpAFic2 substrates were RhoGTPases, 8 and 7, respectively.…”

Section: Fig 2 Global Identification Of Substrates For Vops and Ibpsupporting

confidence: 88%

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Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

Woolery

Luong

et al. 2014

Molecular & Cellular Proteomics

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AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications. Molecular & Cellular

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Section: Fig 2 Global Identification Of Substrates For Vops and Ibpsupporting

confidence: 88%

“…With these efforts combined, four potential new VopS substrates have been identified (SCCA2, NAGK, NME1, and PFKP), though not yet confirmed. These approaches might miss substrates because of temporal and spatial expression or low abundance in cell lysate, poor recognition by the capture molecules or loss during pull-down procedures (12,14).…”

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confidence: 99%

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

Woolery

Luong

et al. 2014

Molecular & Cellular Proteomics

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“…Though target enrichment advanced with the introduction of an antibody raised against AMPylated threonine , tyrosine‐modified peptides, and proteins would escape purification. Another promising step forward was recently presented by a strategy utilizing a functionalized ATP analog that can be trapped by an azide alkyne cycloaddition (also known as CLICK chemistry approach) to enrich for modified proteins . Yet, the modification of ATP goes along with changes in size and electron density of the substrate, which might impair binding to the substrate‐binding site of Fic proteins .…”

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confidence: 99%

An experimental strategy for the identification of AMPylation targets from complex protein samples

et al. 2014

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AMPylation is a posttranslational modification (PTM) that has recently caught much attention in the context of bacterial infections as pathogens were shown to secrete Fic proteins that AMPylate Rho GTPases and thus interfere with host cell signaling processes. Although Fic proteins are widespread and found in all kingdoms of life, only a small number of AMPylation targets are known to date. A major obstacle to target identification is the limited availability of generic strategies allowing sensitive and robust identification of AMPylation events. Here, we present an unbiased MS-based approach utilizing stable isotope-labeled ATP. The ATP isotopes are transferred onto target proteins in crude cell lysates by in vitro AMPylation introducing specific reporter ion clusters that allow detection of AMPylated peptides in complex biological samples by MS analysis. Applying this strategy on the secreted Fic protein Bep2 of Bartonella rochalimae, we identified the filamenting protein vimentin as an AMPylation target that was confirmed by independent assays. Vimentin represents a new class of target proteins and its identification emphasizes our method as a valuable tool to systematically uncover AMPylation targets. Furthermore, the approach can be generically adapted to study targets of other PTMs that allow incorporation of isotopically labeled substrates.

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“…In contrast, there are only scare data about the SELO's function and protein substrates (Sreelatha et al, 2018). Several complementary strategies were introduced to analyze protein AMPylation, including isotopically labeled adenosine probes (Pieles et al, 2014), radioactively labelled adenosine nucleotides, antibodies (Kingdon et al, 1967;Yarbrough et al, 2009), microarrays (Yu and LaBaer, 2015), N 6 -biotin modified ATP (Sreelatha et al, 2018), and N 6 -propargyl adenosine 5'-O-triphosphate (N6pATP) or phosphoramidate (Broncel et al, 2012;Grammel et al, 2011;Kielkowski et al, 2020bKielkowski et al, , 2020a. We have recently developed a chemical proteomic approach, which allows high-throughput screening of protein AMPylation and comparison of the AMPylation levels between different conditions.…”

Section: Introductionmentioning

confidence: 99%

AMPylation is a specific lysosomal protein posttranslational modification in neuronal maturation

Becker

Cappel

et al. 2021

Preprint

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Protein AMPylation is a pervasive posttranslational modification with an emerging role in neurodevelopment. In metazoans the two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques. However, the function of this modification remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. Here, we uncover protein AMPylation as a novel posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chemical proteomics, advanced gel-based separation of modified and unmodified proteins and activity assay, we show that an AMPylated, lysosomal soluble form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation inhibits its catalytic activity. Together, our findings unveil so far unknown lysosomal posttranslational modification, its connection to neuronal differentiation and putatively provide a novel molecular rationale to design of therapeutics for lysosomal storage diseases.

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

Abstract: Tagging protein AMPylation: A new chemical reporter for AMPylation, recently identified as a key post‐translational modification during bacterial infection, is a robust tool for detecting and identifying AMPylated proteins in vitro.

Cited by 17 publications

References 28 publications

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

An experimental strategy for the identification of AMPylation targets from complex protein samples

AMPylation is a specific lysosomal protein posttranslational modification in neuronal maturation

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