Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays.

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.

Chemical reporters for biological discovery.

Functional tools are needed to understand complex biological systems. Here we review how chemical reporters in conjunction with bioorthogonal labeling methods can be used to image and retrieve nucleic acids, proteins, glycans, lipids and other metabolites in vitro, in cells as well as in whole organisms. By tagging these biomolecules, researchers can now monitor their dynamics in living systems and discover specific substrates of cellular pathways. These advances in chemical biology are thus providing important tools to characterize biological pathways and are poised to facilitate our understanding of human diseases.

Chemical reporters for monitoring RNA synthesis and poly(A) tail dynamics.

A versatile "clickable" nucleoside: Metabolic labeling of cells is useful in studying the dynamics of biological molecules. N(6) pA can be utilized by all three mammalian RNA polymerases, as well as poly(A) polymerase. Because of its alkyne modification, RNA labeled with N(6) pA can be visualized and purified by using click chemistry.

A chemical reporter for protein AMPylation.

Protein AMPylation is an emerging post-translational modification, which plays key roles in bacterial pathogenesis and cell biology. Enzymes with AMPylation activity, referred to as AMPylators, have been identified in several bacterial pathogens and eukaryotes. To facilitate the study of this unique modification, we developed an alkynyl chemical reporter for detection and identification of protein AMPylation substrates. Covalent functionalization of AMPylation substrates with the alkynyl reporter in lieu of adenylyl 5'-monophosphate (AMP) allows their subsequent bioorthogonal ligation with azide-fluorescent dyes or affinity enrichment tags. We show that this chemical reporter is transferred by a range of AMPylators onto their cognate protein substrates and allows rapid detection and identification of AMPylated substrates.

Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics.

Proteomic studies have identified a plethora of lysine acetylated proteins in eukaryotes and bacteria. Determining the individual lysine acetyltransferases responsible for each protein acetylation mark is crucial for elucidating the underlying regulatory mechanisms, but has been challenging due to limited biochemical methods. Here, we describe the application of a bioorthogonal chemical proteomics method to profile and identify substrates of individual lysine acetyltransferases. Addition of 4-pentynoyl-coenzyme A, an alkynyl chemical reporter for protein acetylation, to cell extracts, together with purified lysine acetyltransferase p300, enabled the fluorescent profiling and identification of protein substrates via Cu(I)-catalyzed alkyne-azide cycloaddition. We identified several known protein substrates of the acetyltransferase p300 as well as the lysine residues that were modified. Interestingly, several new candidate p300 substrates and their sites of acetylation were also discovered using this approach. Our results demonstrate that bioorthogonal chemical proteomics allows the rapid substrate identification of individual protein acetyltransferases in vitro.

Proteomics analysis of cellular imatinib targets and their candidate downstream effectors.

Inhibition of deregulated protein kinases by small molecule drugs has evolved into a major therapeutic strategy for the treatment of human malignancies. Knowledge about direct cellular targets of kinase-selective drugs and the identification of druggable downstream mediators of oncogenic signaling are relevant for both initial therapy selection and the nomination of alternative targets in case molecular resistance emerges. To address these issues, we performed a proof-of-concept proteomics study designed to monitor drug effects on the pharmacologically tractable subproteome isolated by affinity purification with immobilized, nonselective kinase inhibitors. We applied this strategy to chronic myeloid leukemia cells that express the transforming Bcr-Abl fusion kinase. We used SILAC to measure how cellular treatment with the Bcr-Abl inhibitor imatinib affects protein binding to a generic kinase inhibitor resin and further quantified site-specific phosphorylations on resin-retained proteins. Our integrated approach indicated additional imatinib target candidates, such as flavine adenine dinucleotide synthetase, as well as repressed phosphorylation events on downstream effectors not yet implicated in imatinib-regulated signaling. These included activity-regulating phosphorylations on the kinases Btk, Fer, and focal adhesion kinase, which may qualify them as alternative target candidates in Bcr-Abl-driven oncogenesis. Our approach is rather generic and may have various applications in kinase drug discovery.

Identification of lysine acetyltransferase substrates using bioorthogonal chemical proteomics.

Bioorthogonal chemical proteomics is a valuable method to identify enzyme-specific substrates, a challenging task by traditional biochemical standards. The addition of recombinant enzyme and alkynyl chemical reporter to complex protein mixtures, such as cell lysates, allows the detection and identification of modified substrates. Proteins that have been modified with the chemical reporter can be selectively labeled with fluorescent dyes for detection or affinity tags for biochemical enrichment and subsequent identification by mass spectrometry. Here, we describe the detection and identification of substrates of the lysine acetyltransferase p300 in nuclear extracts using the chemical reporter 4-pentynoyl-CoA.

Cell-selective labeling of bacterial proteomes with an orthogonal phenylalanine amino acid reporter.

Orthogonal amino acid reporters allow the selective labeling of different cell types in heterogeneous populations through the expression of engineered aminoacyl tRNA synthetases. Here, we demonstrate that para-ethynylphenylalanine (PEP) can be used as an orthogonal amino acid reporter for efficient selective labeling of an intracellular bacterial pathogen during infection.

Comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics.

The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na2S2O4-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.