Direct Proximity Tagging of Small Molecule Protein Targets Using an Engineered NEDD8 Ligase

Hill, Z.B.; Pollock, Samuel B.; Zhuang, Min; Wells, James A.

doi:10.1021/jacs.6b06828

Cited by 36 publications

(24 citation statements)

References 28 publications

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“…Complementary approaches that have been developed to offer higher-specificity labeling include NEDDylation and PUP-IT. NEDDylation is based on the fusion of an engineered version of the NEDD8 E2-conjugating enzyme Ubc12 (NEDDylator) to a bait protein or small molecule, with conjugation of the ubiquitin-like NEDD8 protein tag to prey proteins only occurring by direct attack of their lysine ε-amines upon the thioester in the active site of the Bait-NEDDylator 26 . PUP-IT is another prokaryote-derived method, developed to identify membrane protein interactions, in which cells co-express a small protein tag (Pup) with a C-terminal Gly-Gly-Glu domain and the Pup ligase (PafA) fused to a bait protein 27 .…”

Section: Biotin-based Proximity Labeling Approachesmentioning

confidence: 99%

Recent advances in proximity-based labeling methods for interactome mapping

2019

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Proximity-based labeling has emerged as a powerful complementary approach to classic affinity purification of multiprotein complexes in the mapping of protein–protein interactions. Ongoing optimization of enzyme tags and delivery methods has improved both temporal and spatial resolution, and the technique has been successfully employed in numerous small-scale (single complex mapping) and large-scale (network mapping) initiatives. When paired with quantitative proteomic approaches, the ability of these assays to provide snapshots of stable and transient interactions over time greatly facilitates the mapping of dynamic interactomes. Furthermore, recent innovations have extended biotin-based proximity labeling techniques such as BioID and APEX beyond classic protein-centric assays (tag a protein to label neighboring proteins) to include RNA-centric (tag an RNA species to label RNA-binding proteins) and DNA-centric (tag a gene locus to label associated protein complexes) assays.

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Section: Biotin-based Proximity Labeling Approachesmentioning

confidence: 99%

Recent advances in proximity-based labeling methods for interactome mapping

2019

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“…This confirms the direct contact between bait and prey proteins, which ensures neddylation of potential binding partners. This tool has been successfully coupled to SILAC-based quantitative MS to identify small molecule-protein and protein-protein interactions in mammalian cells [122,123]. Peroxidases are oxidoreductases that catalyze redox reactions in the presence of hydrogen peroxide.…”

Section: Proximity Labeling-mass Spectrometry To Study Weak Protein-pmentioning

confidence: 99%

Protein complexes and neighborhoods driving autophagy

Sankar

Dengjel

2020

Autophagy

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Autophagy summarizes evolutionarily conserved, intracellular degradation processes targeting cytoplasmic material for lysosomal degradation. These encompass constitutive processes as well as stress responses, which are often found dysregulated in diseases. Autophagy pathways help in the clearance of damaged organelles, protein aggregates and macromolecules, mediating their recycling and maintaining cellular homeostasis. Protein-protein interaction networks contribute to autophagosome biogenesis, substrate loading, vesicular trafficking and fusion, protein translocations across membranes and degradation in lysosomes. Hypothesis-free proteomic approaches tremendously helped in the functional characterization of protein-protein interactions to uncover molecular mechanisms regulating autophagy. In this review, we elaborate on the importance of understanding protein-protein-interactions of varying affinities and on the strengths of mass spectrometry-based proteomic approaches to study these, generating new mechanistic insights into autophagy regulation. We discuss in detail affinity purification approaches and recent developments in proximity labeling coupled to mass spectrometry, which uncovered molecular principles of autophagy mechanisms. Abbreviations : AMPK: AMP-activated protein kinase; AP-MS: affinity purification-mass spectrometry; APEX2: ascorbate peroxidase-2; ATG: autophagy related; BioID: proximity-dependent biotin identification; ER: endoplasmic reticulum; GFP: green fluorescent protein; iTRAQ: isobaric tag for relative and absolute quantification; MS: mass spectrometry; PCA: protein-fragment complementation assay; PL-MS: proximity labeling-mass spectrometry; PtdIns3P: phosphatidylinositol-3-phosphate; PTM: posttranslational modification; PUP-IT: pupylation-based interaction tagging; RFP: red fluorescent protein; SILAC: stable isotope labeling by amino acids in cell culture; TAP: tandem affinity purification; TMT: tandem mass tag.

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“…An extension to the Nedd8 approach has also been applied to identification of ligand—protein interactions. In this case, SNAP tag, an epitope tag that reacts irreversibly with a benzyl guanidine (Hill et al, 2016), was fused to an engineered Nedd8-conjugating enzyme Ubc12 that is capable of conjugating a biotinylated Nedd8 to proximal proteins. When a benzyl guanidine tag was fused to a small molecule of interest, such as Dasatinib, a Bcr-Abl/Src kinase inhibitor, known binders of Dasatinib were modified by Nedd8.…”

Section: Parametersmentioning

confidence: 99%

Chemical Biology Gateways to Mapping Location, Association, and Pathway Responsivity

2019

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Here we discuss, how by applying chemical concepts to biological problems, methods have been developed to map spatiotemporal regulation of proteins and small-molecule modulation of proteome signaling responses. We outline why chemical-biology platforms are ideal for such purposes. We further discuss strengths and weaknesses of chemical-biology protocols, contrasting them against classical genetic and biochemical approaches. We make these evaluations based on three parameters: occupancy; functional information; and spatial restriction. We demonstrate how the specific choice of chemical reagent and experimental set-up unite to resolve biological problems. Potential improvements/extensions as well as specific controls that in our opinion are often overlooked or employed incorrectly are also considered. Finally, we discuss some of the latest emerging methods to illuminate how chemical-biology innovations provide a gateway toward information hitherto inaccessible by conventional genetic/biochemical means. Finally, we also caution against solely relying on chemical-biology strategies and urge the field to undertake orthogonal validations to ensure robustness of results.

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Direct Proximity Tagging of Small Molecule Protein Targets Using an Engineered NEDD8 Ligase

Cited by 36 publications

References 28 publications

Recent advances in proximity-based labeling methods for interactome mapping

Recent advances in proximity-based labeling methods for interactome mapping

Protein complexes and neighborhoods driving autophagy

Chemical Biology Gateways to Mapping Location, Association, and Pathway Responsivity

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