Tandem Affinity Purification and Mass Spectrometry (TAP‐MS) for the Analysis of Protein Complexes

Adelmant, Guillaume; Garg, Brijesh K.; Tavares, Maria; Card, Joseph D.; Marto, Jarrod A.

doi:10.1002/cpps.84

Cited by 19 publications

(16 citation statements)

References 65 publications

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“…The structure of IER5 suggests that it functions through protein-protein interactions. To identify interacting proteins in an unbiased way, we expressed a tagged form of IER5 in IER5 null I5 cells and performed tandem affinity purification followed by mass spectrometry (Adelmant, Garg, Tavares, Card, & Marto, 2019), which identified the B55a regulatory subunit of PP2A and PP2A scaffolding and catalytic subunits as potential interactors ( Figure 6A; summarized in Table S8). We confirmed these associations by expressing tagged IER5 in I5 cells and tagged B55a in SC2 cells ( Figure 6B).…”

Section: Ier5 Binds B55a/pp2a Complexesmentioning

confidence: 99%

IER5, a DNA-damage response gene, is required for Notch-mediated induction of squamous cell differentiation

Lemieux

Thomas

et al. 2020

Preprint

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Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in longrange enhancers. Among the direct Notch target genes are multiple DNA damage response genes, including IER5, which is regulated by Notch through several enhancer elements. We show that IER5 is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic to PPP2R2A, which encodes the B55a subunit of PP2A, and IER5 interacts with B55a in cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers. ImpactOur findings highlight context-specific crosstalk between Notch, DNA damage response genes, and PP2A, and provide a roadmap for understanding how Notch induces the differentiation of squamous cells.

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Section: Ier5 Binds B55a/pp2a Complexesmentioning

confidence: 99%

IER5, a DNA-damage response gene, is required for Notch-mediated induction of squamous cell differentiation

Lemieux

Thomas

et al. 2020

Preprint

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“…The affinity‐purified protein complexes can be further digested into peptides by trypsin/LysC and identified by quantification of the resulting peptides via mass spectrometry. As the specific interactors are enriched in the bait sample, this method can produce a large amount of information‐rich data that detail protein‐protein interactions in different organisms and biological systems or different conditions and treatment (see Current Protocols article: Adelmant, Garg, Tavares, Card, & Marto, ). Using the putative interactions information, we can further confirm interactions by other binary interaction methods in order to characterize the functions of proteins and provide detailed catalogs of proteins involved in protein complexes and biological processes.…”

Section: Commentarymentioning

confidence: 99%

Rapid Identification of Protein‐Protein Interactions in Plants

et al. 2019

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Enzyme‐enzyme interactions can be discovered by affinity purification mass spectrometry (AP‐MS) under in vivo conditions. Tagged enzymes can either be transiently transformed into plant leaves or stably transformed into plant cells prior to AP‐MS. The success of AP‐MS depends on the levels and stability of the bait protein, the stability of the protein‐protein interactions, and the efficiency of trypsin digestion and recovery of tryptic peptides for MS analysis. Unlike in‐gel‐digestion AP‐MS, in which the gel is cut into pieces for several independent trypsin digestions, we uses a proteomics‐based in‐solution digestion method to directly digest the proteins on the beads following affinity purification. Thus, a single replicate within an AP‐MS experiment constitutes a single sample for LC‐MS measurement. In subsequent data analysis, normalized signal intensities can be processed to determine fold‐change abundance (FC‐A) scores by use of the SAINT algorithm embedded within the CRAPome software. Following analysis of co‐sublocalization of “bait” and “prey,” we suggest considering only the protein pairs for which the intensities were more than 2% compared with the bait, corresponding to FC‐A values of at least four within‐biological replicates, which we recommend as minimum. If the procedure is faithfully followed, experimental assessment of enzyme‐enzyme interactions can be carried out in Arabidopsis within 3 weeks (transient expression) or 5 weeks (stable expression). © 2019 The Authors. Basic Protocol 1: Gene cloning to the destination vectors Alternate Protocol: In‐Fusion or Gibson gene cloning protocol Basic Protocol 2: Transformation of baits into the plant cell culture or plant leaf Basic Protocol 3: Affinity purification of protein complexes Basic Protocol 4: On‐bead trypsin/LysC digestion and C18 column peptide desalting and concentration Basic Protocol 5: Data analysis and quality control

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“…Protein complexes isolated by tandem affinity purification (39) were directly processed in solution: Cysteine residues were first reduced with 10 mM dithiothreitol for 30 minutes at 56°C in the presence of 0.1% RapiGest SF (Waters, Milford, MA) and then alkylated with 22.5 mM iodoacetamide for 20 minutes at room temperature in the dark. Proteins were digested overnight at 37°C using 2.5 micrograms of trypsin after adjusting the pH to 8.0 with Tris.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling

et al. 2019

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Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.

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Tandem Affinity Purification and Mass Spectrometry (TAP‐MS) for the Analysis of Protein Complexes

Cited by 19 publications

References 65 publications

IER5, a DNA-damage response gene, is required for Notch-mediated induction of squamous cell differentiation

IER5, a DNA-damage response gene, is required for Notch-mediated induction of squamous cell differentiation

Rapid Identification of Protein‐Protein Interactions in Plants

Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling

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