Conformational exchange at a C2H2 zinc-binding site facilitates redox sensing by the PML protein.

The promyelocytic leukemia protein, PML, plays a vital role in the cellular response to oxidative stress; however, the molecular mechanism of its action remains poorly understood. Here, we identify redox-sensitive sites of PML. A molecule of PML is cysteine-rich and contains three zinc-binding domains including RING, B-box1, and B-box2. Using in vitro assays, we have compared the sensitivity of the isolated RING and B-box1 domains and shown that B-box1 is more sensitive to oxidation. NMR studies of PML dynamics showed that one of the Zn-coordination sites within the B-box1 undergoes significant conformational exchange, revealing a hotspot for exposure of reactive cysteines. In agreement with the in vitro data, enhancement of the B-box1 Zn-coordination dynamics led to more efficient recruitment of PML into PML nuclear bodies in cells. Overall, our results suggest that the increased sensitivity of B-box1 to oxidative stress makes this domain an important redox-sensing component of PML.

The B-box1 domain of PML mediates SUMO E2-E3 complex formation through an atypical interaction with UBC9.

The small, ubiquitin-like modifier SUMO is covalently attached to substrates by the enzyme UBC9. SUMO conjugation of substrates often requires an E3 ligase, which ensures substrate specificity by simultaneously binding UBC9 and the substrate. E3 SUMO ligases commonly use a RING domain to engage UBC9. The Promyelocytic Leukemia protein (PML) has been implicated as a probable SUMO ligase. Although PML does contain a RING domain, which is expected to recruit UBC9, we demonstrate that PML RING does not bind UBC9 in vitro. Instead, we show that isolated PML B-box1 possesses UBC9-binding activity and map the B-box1 binding site on UBC9. This site also binds the upstream E1 enzyme that transfers SUMO to UBC9. The overlap of these two binding sites suggests that UBC9 cannot interact with its E1 and E3 partners simultaneously. Furthermore, we present a model of the PML dimer that supports the accessibility of B-box1 for UBC9 binding in the context of the full-length PML.

Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60.

Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7) is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-generation USP7 inhibitor with improved potency and selectivity. P217564 selectively targets the catalytic cleft of USP7 and modifies its active site cysteine (C223) by forming a covalent adduct. Irreversible inhibition of USP7 results in durable downstream biological responses in cells, including down-regulation of Tip60 and consequent impairment of Treg suppressive function. In addition, we demonstrate that both USP7 and various USP7 substrates are subjected to Lys48-mediated ubiquitin modification, consistent with increased proteasomal degradation of these proteins because of USP7 inhibition.