Targeted recruitment of USP15 enhances CTLA4 surface levels and restricts its degradation.

Abstract

Induced protein proximity offers powerful new routes to modulate protein fate. While proteolysis-targeting chimeras (PROTACs) promote degradation through E3 ligase recruitment, the converse principle--targeted protein stabilisation or enhancement via deubiquitylase (DUB) recruitment--is only beginning to emerge. The immune checkpoint receptor CTLA4, whose deficiency causes severe autoimmunity, undergoes rapid ubiquitin-dependent lysosomal degradation, making it one of the most short-lived transmembrane proteins. Using an inducible "RapTag" system, which brings together tagged proteins through rapalog-mediated FKBP-FRB dimerisation, we show that enforced proximity to the broad-specificity DUB USP15 markedly increases total and cell-surface CTLA4 levels. Controlled expression of wild-type or catalytically inactive USP15 in isogenic cell lines revealed a clear requirement for DUB activity. The elevation of CTLA4 at the plasma membrane exceeded that of the total cellular pool, consistent with a diversion from ubiquitin-driven lysosomal sorting towards recycling. This easily adaptable platform enables systematic testing of DUB-substrate combinations that informs rational ENhancement TArgeting Chimera (ENTAC) design for downstream drug discovery efforts and targeted protein rescue in therapeutic contexts. Significance statementBy showing that DUB proximity redirects CTLA4 from lysosomal degradation to the plasma membrane, this work defines the mechanistic foundation for Enhancement Targeting Chimeras (ENTACs) that stabilise and correctly reposition unstable proteins.