APEX2‐based Proximity Labeling of Atox1 Identifies CRIP2 as a Nuclear Copper‐binding Protein that Regulates Autophagy Activation

Abstract

Mammalian cell nuclei contain copper,a nd cancer cells are knownt oa ccumulate aberrantly high copper levels, yet the mechanisms underlying nuclear accumulation and coppersbroader functional significance remain poorly understood. Here,b yc ombining APEX2-based proximity labeling focused on the copper chaperone Atox1 with mass spectrometry we identified ap reviously unrecognized nuclear copper binding protein, Cysteine-rich protein 2(CRIP2), that interacts with Atox1 in the nucleus.Weshowthat Atox1 transfers copper to CRIP2, whichi nduces ac hange in CRIP2'ss econdary structure that ultimately promotes its ubiquitin-mediated proteasomal degradation. Finally,w ed emonstrate that depletion of CRIP2-as well as copper-induced CRIP2 degradationelevates ROSl evels and activates autophagy in H1299 cells. Thus,o ur study establishes that CRIP2 as an autophagic suppressor protein and implicates CRIP2-mediated copper metabolism in the activation of autophagy in cancer cells.

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