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Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified lysosomal cholesterol signaling (LYCHOS, previously annotated as G protein-coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic target of rapamycin complex 1 (mTORC1). Cholesterol bound to the amino-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation. At high cholesterol concentrations, LYCHOS bound to the GATOR1 complex, a guanosine triphosphatase (GTPase)-activating protein for the Rag GTPases, through a conserved cytoplasm-facing loop. By sequestering GATOR1, LYCHOS promotes cholesterol- and Rag-dependent recruitment of mTORC1 to lysosomes. Thus, LYCHOS functions in a lysosomal pathway for cholesterol sensing and couples cholesterol concentrations to mTORC1-dependent anabolic signaling.

Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73. To identify all the binding sites of neurosteroids in VDAC1, we apply photo-affinity labeling using two sterol-based photolabeling reagents with complementary photochemistry: 5α-6-AziP which contains an aliphatic diazirine, and KK200 which contains a trifluoromethyl-phenyldiazirine (TPD) group. 5α-6-AziP and KK200 photolabel multiple residues within an E73 pocket confirming the presence of this site and mapping sterol orientation within this pocket. In addition, KK200 photolabels four other sites consistent with the finding that VDAC1 co-purifies with five cholesterol molecules. Both allopregnanolone and cholesterol competitively prevent photolabeling at E73 and three other sites indicating that these are common sterol binding sites shared by both neurosteroids and cholesterol. Binding at the functionally important residue E73 suggests a possible role for sterols in regulating VDAC1 signaling and interaction with partner proteins.

Background and Purpose: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady‐state binding affinities of various neurosteroids for specific sites on the GABAA receptor. Experimental Approach: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17‐methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC‐α1GABAAR, a chimeric receptor containing transmembrane domains of the α1‐GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. Key Results: Allopregnanolone (3α‐OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi‐allopregnanolone (3β‐OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site‐specific probe for the intersubunit site. The affinity and site‐specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. Conclusions and Implications: The affinity and specificity of neurosteroid binding to two sites in the ELIC‐α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate‐reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites. [ABSTRACT FROM AUTHOR]

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue.

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABAA receptors. Interestingly, the PAM effects of ALLO are strongly enantioselective, whereas those of PREG are not. This study was aimed at determining the basis for this difference in enantioselectivity. The oocyte electrophysiology studies showed that ent-ALLO potentiates GABA-elicited currents in α1β3 GABAA receptors with lower potency and efficacy than ALLO, PREG or ent-PREG. The small PAM effect of ent-ALLO was prevented by the α1(Q242L) mutation in the intersubunit neurosteroid binding site between the β3 and α1 subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that ent-ALLO binds weakly to the β3-α1 intersubunit binding site in comparison to ALLO, PREG and ent-PREG. Rigid body docking predicted that ent-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or ent-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and ent-ALLO binding to the α1 or β3 intrasubunit binding sites that also mediate neurosteroid modulation of GABAA receptors. The results demonstrate that differential binding of ent-ALLO and ent-PREG to the β3-α1 intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG. [ABSTRACT FROM AUTHOR]

19F-NMR T2 relaxation was employed to study whether other volatile anesthetics besides isoflurane bind to the same fatty acid-displaceable regions on serum albumin, and whether they differ in their attraction to these regions. Three other flurinated volatile anesthetics, halothane, methoxyflurane and sevoflurane, bind with differing attraction to fatty-acid displaceable regions on serum albumin, and impair isoflurane binding competitively. Several different volatile anesthetics can compete for binding to particular regions on a rotein.

The ligand-binding interaction between the anesthetic isoflurane and bovine serum albumin (BSA) was characterized using 19F-nuclear magnetic resonance transverse relaxation. Results indicate that there is a rapid exchange of the anesthetic between BSA and the medium. Furthermore, the investigation provided direct evidence that particular proteins in certain conformations possess a binding site for fluorinated anesthetics, providing a method sensitive enough to study the ligand-binding interaction.