In addition to two well-recognized proteasome subtypes—constitutive proteasomes and immunoproteasomes—mounting evidence also suggests the existence of intermediate proteasome subtypes containing unconventional mixtures of catalytic subunits. Although they appear to play unique biological roles, the lack of practical methods for detecting distinct proteasome subtypes has limited functional investigations. Here, we report the development of activity-based probes that crosslink two catalytic subunits within intact proteasome complexes. Identification of the crosslinked subunit pairs provides direct evidence of the catalytic subunit composition of proteasomes. Using these probes, we found that U266 multiple myeloma cells contain intermediate proteasomes comprising both β1i and β2, but not β1 and β2i, consistent with previous findings with other cell types. Our bifunctional probes can be utilized in functional investigations of distinct proteasome subtypes in various biological settings.

Estrogen receptor-α (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, hormone-sensitive ER-positive cancer cells eventually develop resistance to ER antagonists. It has been shown that in most of these resistant tumor cells, the ER is expressed and continues to regulate tumor growth. Recent studies indicate that tamoxifen initially acts as an antagonist, but later functions as an ER agonist, promoting tumor growth. This suggests that targeted ER degradation may provide an effective therapeutic approach for breast cancers, even those that are resistant to conventional therapies. With this in mind, we previously demonstrated that proteolysis targeting chimeras (PROTACs) effectively induce degradation of the ER as a proof-of-concept experiment. Herein we further refined the PROTAC approach to target the ER for degradation. The ER-targeting PROTACs are composed of an estradiol on one end and a hypoxia-inducing factor 1α (HIF-1α)-derived synthetic pentapeptide on the other. The pentapeptide is recognized by an E3 ubiquitin ligase called the von Hippel Lindau tumor suppressor protein (pVHL), thereby recruiting the ER to this E3 ligase for ubiquitination and degradation. Specifically, the pentapeptide is attached at three different locations on estradiol to generate three different PROTAC types. With the pentapeptide linked through the C7α position of estradiol, the resulting PROTAC shows the most effective ER degradation and highest affinity for the estrogen receptor. This result provides an opportunity to develop a novel type of ER antagonist that may overcome the resistance of breast tumors to conventional drugs such as tamoxifen and fulvestrant (Faslodex).