ABSTRACT
Pregnane X receptor (PXR) is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes [e.g., cytochrome P450 3A4 (CYP3A4)] and transporters. Although the regulation of PXR target genes is well-characterized, less is known about the regulation of PXR protein level. By screening an RNAi library, we identified the F-box-only protein 44 (FBXO44) as a novel E3 ligase for PXR. PXR abundance increases upon knockdown of FBXO44, and, inversely, decreases upon overexpression of FBXO44. Further analysis revealed that FBXO44 interacts with PXR, leading to its ubiquitination and proteasomal degradation, and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction. In summary, FBXO44 regulates PXR protein abundance, which has downstream consequences for CYP3A4 levels and drug-drug interactions. The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3 ubiquitin ligase activities will affect PXR-mediated drug metabolism.
ABSTRACT
Intracellular protein trafficking and sorting are extremely arduous in endocrine and neuroendocrine cells, which synthesize and secrete on-demand substantial quantities of proteins. To ensure that neuroendocrine secretion operates correctly, each step in the secretion pathways is tightly regulated and coordinated both spatially and temporally. At the trans-Golgi network (TGN), intrinsic structural features of proteins and several sorting mechanisms and distinct signals direct newly synthesized proteins into proper membrane vesicles that enter either constitutive or regulated secretion pathways. Furthermore, this anterograde transport is counterbalanced by retrograde transport, which not only maintains membrane homeostasis but also recycles various proteins that function in the sorting of secretory cargo, formation of transport intermediates, or retrieval of resident proteins of secretory organelles. The retromer complex recycles proteins from the endocytic pathway back to the plasma membrane or TGN and was recently identified as a critical player in regulated secretion in the hypothalamus. Furthermore, melanoma antigen protein L2 (MAGEL2) was discovered to act as a tissue-specific regulator of the retromer-dependent endosomal protein recycling pathway and, by doing so, ensures proper secretory granule formation and maturation. MAGEL2 is a mammalian-specific and maternally imprinted gene implicated in Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. In this review, we will briefly discuss the current understanding of the regulated secretion pathway, encompassing anterograde and retrograde traffic. Although our understanding of the retrograde trafficking and sorting in regulated secretion is not yet complete, we will review recent insights into the molecular role of MAGEL2 in hypothalamic neuroendocrine secretion and how its dysregulation contributes to the symptoms of Prader-Willi and Schaaf-Yang patients. Given that the activation of many secreted proteins occurs after they enter secretory granules, modulation of the sorting efficiency in a tissue-specific manner may represent an evolutionary adaptation to environmental cues.
ABSTRACT
We previously reported a specific inverse agonist (SPA70) of the nuclear receptor pregnane X receptor (PXR). However, derivatization of SPA70 yielded only agonists and neutral antagonists, suggesting that inverse agonism of PXR is difficult to achieve. Therefore, we sought to design proteolysis targeting chimeras (PROTACs) aimed at inducing PXR degradation. Conjugation of a SPA70 derivative to ligands of the E3 substrate receptor cereblon (CRBN) resulted in one molecule, SJPYT-195, that reduced PXR protein level in an optimized degradation assay described here. Further analysis revealed that SJPYT-195 was a molecular glue degrader of the translation termination factor GSPT1 and that GSPT1 degradation resulted in subsequent reduction of PXR protein. GSPT1 has recently gained interest as an anticancer target, and our results give new insights into chemical determinants of drug-induced GSPT1 degradation. Additionally, we have developed assays and cell models for PXR degrader discovery that can be applied to additional protein targets.
ABSTRACT
The melanoma antigen (MAGE) proteins all contain a MAGE homology domain. MAGE genes are conserved in all eukaryotes and have expanded from a single gene in lower eukaryotes to â¼40 genes in humans and mice. Whereas some MAGEs are ubiquitously expressed in tissues, others are expressed in only germ cells with aberrant reactivation in multiple cancers. Much of the initial research on MAGEs focused on exploiting their antigenicity and restricted expression pattern to target them with cancer immunotherapy. Beyond their potential clinical application and role in tumorigenesis, recent studies have shown that MAGE proteins regulate diverse cellular and developmental pathways, implicating them in many diseases besides cancer, including lung, renal, and neurodevelopmental disorders. At the molecular level, many MAGEs bind to E3 RING ubiquitin ligases and, thus, regulate their substrate specificity, ligase activity, and subcellular localization. On a broader scale, the MAGE genes likely expanded in eutherian mammals to protect the germline from environmental stress and aid in stress adaptation, and this stress tolerance may explain why many cancers aberrantly express MAGEs Here, we present an updated, comprehensive review on the MAGE family that highlights general characteristics, emphasizes recent comparative studies in mice, and describes the diverse functions exerted by individual MAGEs.
