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Comparative_Interaction_Proteomics_GFP-tagged_MAGEC2_HEK293T

ABSTRACT: 293T (Human embryonic kidney cells-ATCC CRL-3216) cells were plated at 500,000 cells per 10 cm dish and grown for 48 hours. Cells were then transfected with GFP-MAGEC1 WT or GFP-MAGEC1 L318D in a 3:1 ratio of PEI (polyethylenimine) to DNA. Cells were harvested after 48 hours and lysed in 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitor (cOmpleteâ„¢, Mini, EDTA-free (Roche)). Cells were incubated for 25 minutes on ice and then sonicated for 5 minutes at 4Â°C. Cells were centrifuged at 14,000 rpm for 20 minutes at 4 Â°C. 500 ug lysate was then added to GFP-TrapÂ® Agarose resin (ChromoTek) and samples were incubated rotating at 4Â°C for 2 hours. Following washing of the beads with 50 mM Tris pH 7.5, 150 mM NaCl, proteins were eluted by the addition of 2x Laemmli buffer and boiled for 10 minutes at 95 Â°C. Samples were spun down and the supernatants were sent for mass spectrometry analysis as described below. Experiments were conducted in triplicate.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Frank Stein

LAB HEAD: Sagar Bhogaraju

PROVIDER: PXD047849 | Pride | 2024-02-20

REPOSITORIES: Pride

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			Action	DRS
	FragPipe_workflow.workflow	Other
	UP000005640_HomoSapiens_ID9606_20594entries_26102022_dl11012023.fasta	Fasta
	Zelda_230412_P2593_MR_SG_IP_TMT9_fr1.raw	Raw
	Zelda_230412_P2593_MR_SG_IP_TMT9_fr2.raw	Raw
	Zelda_230412_P2593_MR_SG_IP_TMT9_fr3.raw	Raw

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Project description:Ubiquitination of proteins is central to protein homeostasis and other cellular processes including DNA repair, vesicular transport, cell-division etc. The process of ubiquitination is conserved from yeast to humans and is carried out by the sequential action of three enzymes: E1, E2 and E3. There are an estimated >600 E3 ligases in humans that execute ubiquitination of specific target proteins in a spatio-temporal manner to elicit desired signaling effects. Here, we developed a simple, proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. Our method exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2~Ub. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich the bona fide substrates and potential new substrates of the ligase using a one-step Streptavidin pulldown under denaturing conditions. As proof-of-principle, we applied our method, which we named Ub-POD, to the RING E3 ligase Rad18. Rad18 ubiquitinates DNA-sliding clamp PCNA upon UV-induced DNA damage. We identified PCNA and several other critical players in the DNA damage repair pathway in a single Ub-POD experiment. We further discovered an unintended but important application of Ub-POD, where we were able to pin down the cellular localization of Rad18- mediated ubiquitination to the damaged DNA nuclear puncta through Streptavidin immunofluorescence. We also applied our method to TRAF6, another RING ubiquitin ligase involved in TNF signaling, successfully identifying known and potentially new substrates. Finally, we adapted our method to the U-box type E3 ubiquitin ligase CHIP to demonstrate that we can identify substrates of two major classes of mammalian ubiquitin ligases. We anticipate that our method and principle could be widely adapted to all classes of ubiquitin ligases to identify substrates and localize the cellular site(s) of ubiquitination.

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Comparative_Interaction_Proteomics_GFP-tagged_MAGEC2_HEK293T

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