Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitin is an evolutionarily conserved protein of 76 amino acids^1,2,3. Ubiquitin covalently binds lysine residues on target proteins through cascades involving activating (E1), conjugating (E2), and ligase (E3) enzymes. Ubiquitin is first activated by the E1 enzyme and is then transferred to the E2 conjugating enzymes. Subsequently, E3 ubiquitin ligases interact with both ubiquitin-loaded E2 enzymes and substrate proteins and mediate the formation of an isopeptide bond between the C-terminal of ubiquitin and a lysine residue in the substrate^1,2,3,4,5. Ubiquitination involves the attachment of ubiquitin moieties to lysine residues on substrate proteins or to itself, leading to protein monoubiquitination or polyubiquitination. This ubiquitination process occurs intracellularly in eukaryotes and regulates a large variety of biological processes. Ubiquitination results in the degradation of substrate proteins via the ubiquitin-proteasome system^1,2,3,4,5. In addition, ubiquitination modulates protein subcellular localization, protein complex formation, and protein trafficking in cells^3,5. Ubiquitin moieties ligated to substrate proteins can be removed by deubiquitinating enzymes (DUBs)^6,7. Notably, the different ways in which ubiquitin chains are assembled provide a myriad of means to regulate various biological processes^1,5. The exact roles of ubiquitination in regulating substrate protein function remain incompletely understood till now. The purification of ubiquitinated proteins contributes to the elucidation of the effects of protein ubiquitination on a variety of cellular processes.

The p53 protein is one of the most important tumor suppressor proteins and exhibits genetic mutations or inactivation in almost all human cancers^8,9,10,11. p53 stability and activity are delicately regulated in vivo by posttranslational modifications, including ubiquitination, phosphorylation, acetylation, and methylation^12,13. The p53 protein has a short half-life ranging from 6 min to 40 min in various cells, which results mainly from its polyubiquitination and subsequent proteasomal degradation^10,12. Mouse double minute 2 (Mdm2) is an E3 ubiquitin ligase of p53 that binds to the N-terminus of p53 to inhibit its transcriptional activity^12,14,15. Mdm2 promotes the polyubiquitination and proteasomal degradation of p53 to control its stability and induces monoubiquitination of p53 to facilitate its nuclear export^12,14,15,16. Here, Mdm2-mediated p53 ubiquitination is used as an example to introduce a method for the purification of ubiquitinated proteins from mammalian cells in detail. The regulators that influence the ubiquitination status of target proteins can be identified using this in vivo ubiquitination assay when they are overexpressed or knocked down/knocked out in mammalian cells. In addition, ubiquitinated proteins can be used as substrates for in vitro deubiquitination assay. A high-throughput screening can be performed to identify specific DUBs for target proteins by incubating ubiquitinated substrates with individual DUBs. Ubiquitinated proteins may act as a scaffold to recruit downstream signaling proteins in cells. A ubiquitinated target protein complex can be purified by sequential immunoprecipitation under native purification conditions and identified by mass-spectrometry. The current protocol can be extensively used to investigate the cellular proteins regulated by ubiquitination.

Several methods have been established to purify ubiquitinated proteins, which include the use of affinity-tagged ubiquitin, ubiquitin antibodies, ubiquitin-binding proteins, and isolated ubiquitin-binding domains (UBDs)¹⁷. Here, we provide a protocol using affinity-tagged ubiquitin as a mediator to purify ubiquitinated proteins in mammalian cells. The use of poly-His-tagged ubiquitin offers advantages over the other methods. Ubiquitinated proteins are purified in the presence of strong denaturants, which reduces non-specific binding to nickel-charged resin by linearizing cellular proteins and disrupting protein-protein interactions. In contrast, the use of ubiquitin antibodies, ubiquitin-binding proteins, and isolated UBDs as mediators cannot effectively exclude binding partners from target protein because purification needs to be performed under less stringent conditions. Moreover, purification may also lead to increased binding of unrelated proteins using these mediators. In addition, there is a binding propensity for various ubiquitin linkage types as well as mono- and poly-ubiquitination by ubiquitin-binding proteins or isolated UBDs¹⁷. The use of poly-His-tagged ubiquitin contributes to pull down all cellular ubiquitinated proteins. Alternatively, the use of commercially available anti-Flag or anti-HA antibody-conjugated agarose make it easier to immunoprecipitate large-scale Flag- or HA-tagged target proteins under nondenaturing conditions. A second purification step, for example, by nickel-charged resin targeting poly-His-tagged ubiquitin, can be used to acquire ubiquitinated target proteins with a high purity for downstream experiments. Notably, an epitope tagging purification strategy can be adapted when a specific antibody cannot be acquired to immunoprecipitate target proteins effectively. Finally, purification of ubiquitinated proteins in mammalian cells, in comparison with purification in vitro, retains the ubiquitin linkage mode of target proteins under more physiological conditions.