ABSTRACT
Hedgehog (Hh) signaling pathway plays a pivotal role in embryonic development. Hh binding to Patched1 (PTCH1) derepresses Smoothened (SMO), thereby activating the downstream signal transduction. Covalent SMO modification by cholesterol in its cysteine-rich domain (CRD) is essential for SMO function. SMO cholesterylation is a calcium-accelerated autoprocessing reaction, and STIM1-ORAI1-mediated store-operated calcium entry promotes cholesterylation and activation of endosome-localized SMO. However, it is unknown whether the Hh-PTCH1 interplay regulates the activity of the endoplasmic reticulum (ER)-localized SMO. Here, we found that PTCH1 inhibited the COPII-dependent export of SMO from the ER, whereas Hh promoted this process. The RRxWxR amino acid motif in the cytosolic tail of SMO was essential for COPII recognition, ciliary localization, and signal transduction activity. Hh and PTCH1 regulated cholesterol modification of the ER-localized SMO, and SMO cholesterylation accelerated its exit from ER. The GRAMD1/ASTER sterol transport proteins facilitated cholesterol transfer to ER from PM, resulting in increased SMO cholesterylation and enhanced Hh signaling. Collectively, we reveal a regulatory role of GRAMD-mediated cholesterol transport in ER-resident SMO maturation and Hh signaling.
Subject(s)
Calcium , Hedgehog Proteins , Biological Transport , Calcium/metabolism , Cholesterol/metabolism , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/physiology , Smoothened Receptor/genetics , Smoothened Receptor/metabolism , Membrane Proteins/metabolismABSTRACT
Inducible degrader of the low-density lipoprotein receptor (IDOL) is an E3 ubiquitin ligase mediating degradation of low-density lipoprotein (LDL) receptor (LDLR). IDOL also controls its own stability through autoubiquitination, primarily at lysine 293. Whether IDOL may undergo other forms of posttranslational modification is unknown. In this study, we show that IDOL can be modified by small ubiquitin-like modifier 1 at the K293 residue at least. The SUMOylation of IDOL counteracts its ubiquitination and augments IDOL protein levels. SUMOylation and the associated increase of IDOL protein are effectively reversed by SUMO-specific peptidase 1 (SENP1) in an activity-dependent manner. We further demonstrate that SENP1 affects LDLR protein levels by modulating IDOL. Overexpression of SENP1 increases LDLR protein levels and enhances LDL uptake in cultured cells. On the contrary, loss of SENP1 lowers LDLR levels in an IDOL-dependent manner and reduces LDL endocytosis. Collectively, our results reveal SUMOylation as a new regulatory posttranslational modification of IDOL and suggest that SENP1 positively regulates the LDLR pathway via deSUMOylation of IDOL and may therefore be exploited for the treatment of cardiovascular disease.
Subject(s)
Cysteine Endopeptidases/metabolism , Receptors, LDL/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Line , Humans , Protein Processing, Post-Translational , Sumoylation , UbiquitinationABSTRACT
The hedgehog (Hh) signaling pathway has long been a hotspot for anti-cancer drug development due to its important role in cell proliferation and tumorigenesis. However, most clinically available Hh pathway inhibitors target the seven-transmembrane region (7TM) of smoothened (SMO), and the acquired drug resistance is an urgent problem in SMO inhibitory therapy. Here, we identify a sterol analog Q29 and show that it can inhibit the Hh pathway through binding to the cysteine-rich domain (CRD) of SMO and blocking its cholesterylation. Q29 suppresses Hh signaling-dependent cell proliferation and arrests Hh-dependent medulloblastoma growth. Q29 exhibits an additive inhibitory effect on medulloblastoma with vismodegib, a clinically used SMO-7TM inhibitor for treating basal cell carcinoma (BCC). Importantly, Q29 overcomes resistance caused by SMO mutants against SMO-7TM inhibitors and inhibits the activity of SMO oncogenic variants. Our work demonstrates that the SMO-CRD inhibitor can be a new way to treat Hh pathway-driven cancers.
Subject(s)
Cell Proliferation , Hedgehog Proteins , Medulloblastoma , Signal Transduction , Smoothened Receptor , Sterols , Smoothened Receptor/antagonists & inhibitors , Smoothened Receptor/metabolism , Hedgehog Proteins/metabolism , Hedgehog Proteins/antagonists & inhibitors , Humans , Signal Transduction/drug effects , Animals , Mice , Cell Proliferation/drug effects , Sterols/chemistry , Sterols/pharmacology , Sterols/metabolism , Medulloblastoma/drug therapy , Medulloblastoma/metabolism , Medulloblastoma/pathology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Molecular Structure , Cholesterol/metabolismABSTRACT
Hedgehog (Hh) is a morphogen that binds to its receptor Patched 1 and activates Smoothened (SMO), thereby governing embryonic development and postnatal tissue homeostasis. Cholesterol can bind and covalently conjugate to the luminal cysteine-rich domain (CRD) of human SMO at the D95 residue (D99 in mouse). The reaction mechanism and biological function of SMO cholesterylation have not been elucidated. Here, we show that the SMO-CRD undergoes auto-cholesterylation which is boosted by calcium and involves an intramolecular ester intermediate. In cells, Hh stimulation elevates local calcium concentration in the SMO-localized endosomes through store-operated calcium entry. In addition, we identify the signaling-incompetent SMO D95E mutation, and the D95E mutant SMO can bind cholesterol but cannot be modified or activated by cholesterol. The homozygous SmoD99E/D99E knockin mice are embryonic lethal with severe developmental delay, demonstrating that cholesterylation of CRD is required for full-length SMO activation. Our work reveals the unique autocatalytic mechanism of SMO cholesterylation and an unprecedented role of calcium in Hh signaling.