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Proteomic Profiling of Potential E6AP Substrates via Ubiquitin-based Photo-Crosslinking Assisted Affinity Enrichment

ABSTRACT: E6AP/UBE3A, the founding member of HECT-type (Homologous to E6AP C-terminus) E3 ligases, is implicated in human papillomaviruses (HPV)-mediated cervical cancer and neurodevelopmental disorders like Angelman and Dup15q syndromes. To elucidate the function of E3 ligases, knowledge about their substrates is crucial. Their identification, however, is challenging due to complex cross-reactivities between E2, E3 and their substrates. To address this, we developed a novel ubiquitin-based photo labeling approach for HECT-type ligases. Our method modifies ubiquitin with a diazirine moiety and attaches it to E6AP's active site using the ubiquitination cascade and a Cys to Lys mutation. The resulting E6AP-Ub conjugate enables substrate trapping via photoaffinity labeling and proteomic profiling. We validated this approach through literature comparison and identified Ub C-terminal hydrolase 7 (Uchl5/Uch37) as a new, proteasome-independent ubiquitination target of E6AP. This method expands the toolbox for substrate identification, potentially providing deeper insights into E6AP-related cellular processes and other HECT-type E3 ligases.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell

SUBMITTER: Julian Schuck

LAB HEAD: Andreas Marx

PROVIDER: PXD055649 | Pride | 2025-01-27

REPOSITORIES: pride

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	Q2304_JS_230426_10_256.raw	Raw
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