Blog

By Yu-Shiuan Cheng, Tianyi Zhang, Xiang Ma, Sarida Pratuangtham, Grace C. Zhang, Alexander A. Ondrus, Amirhossein Mafi, Brett Lomenick, Jeffrey J. Jones, and Alison E. Ondrus

Excerpt from the author manuscript published in Nature Chemical Biology 17, 1271–1280 (19 November 2021 DOI: https://doi.org/10.1038/s41589-021-00907-2

Editor’s Highlights

• Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease.
• OHC targets have functional roles in signal transduction, protein trafficking, and membrane lipid metabolism, and we identify specific processes in immune response and cancer.
• 20(S)-hydroxycholesterol (20(S)-OHC), is an agonist of the developmental signaling protein Smoothened (Smo), a key transmembrane protein that is a key component of the hedgehog signaling pathway.
• The receptor Tmem97 is a regio-and stereoselective 20(S)-OHC target that links OHC binding to cholesterol homeostasis.
• Ligation of Tmem97 by 20(S)-OHC may influence its association with Niemann-Pick C1 (NPC1), associated with mitochondrial dysfunction and mTOR suppression.
• OHC-coordinated processes targeting TANK binding kinase 1 (TBK1) in KRAS-mutant lung adenocarcinoma cells is synthetically lethal and represent an avenue for the treatment of refractory KRAS mutant cancers. 

Abstract

Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. In this manuscript, we present a structurally precise chemoproteomics probe for the biologically active molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalogue consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20(S)-OHC is a chemo-, regio-, and stereoselective ligand for the protein Tmem97 (the σ2 receptor), enabling us to reconstruct the 20(S)-OHC:Tmem97 binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of metabolite activity and identify actionable targets for molecular therapy.

Introduction

Cholesterol is the most complex metabolite produced de novo by humans.¹ It serves as a substrate for the biosynthesis of an edifice of signaling molecules, including steroid hormones, glucocorticoids, bile acids, and vitamin D.² Over one hundred human enzymes are dedicated to modifying cholesterol structure, whereas no catabolic enzymes are available to derive energy from its breakdown. Instead, the human body expresses a multitude of cholesterol metabolite receptors that control steroidogenesis, respiration, gut health, vision, and a repertoire of other essential processes. The gateway from cholesterol to all of these metabolites is a class of intermediates known as oxysterols (OHCs): specific mono-, di-, and polyhydroxylated cholesterol molecules that are barcoded by the configuration of their oxygen atoms.^3,4 Although they serve as precursors to hundreds of metabolite structures, oxysterols are maintained at fleetingly low concentrations in the body and are rapidly converted to more metabolically stable products.⁵ A growing body of evidence has revealed that these ephemeral intermediates also function as sensitive, highly potent signaling molecules in their own right, coordinating critical processes such as neurotransmission, developmental signaling, and the innate immune response, where temporal control and specificity are paramount. Despite the fact that the chemical structures of OHCs directly reflect their receptor preferences, only a fraction of the proteins that interact with these molecules is known. To tap into their signaling functions and trace the circuits that regulate their activity, chemoproteomic technologies to fingerprint the cellular targets of specific OHC structures are required.

An accurate portrait of proteome-wide OHC interactions in live cells requires that chemoproteomics probes preserve the oxygenation pattern of the natural congener as well as physiochemical properties for native membrane partitioning.⁶ In this work, we describe our design, synthesis, and application of a structurally precise photoaffinity probe for 20(S)-hydroxycholesterol (20(S)-OHC), an agonist of the developmental signaling protein Smoothened.^7,8 Using this probe, we perform an unbiased analysis of live-cell 20(S)-OHC targets in the membrane proteome of mammalian cells. Our data reveals over 100 enriched 20(S)-OHC interactors that cluster into discrete molecular functions. We elucidate a set of protein interactors that competably engage 20(S)-OHC and show that the orphan receptor Tmem97 is a regio-and stereoselective 20(S)-OHC target that links OHC binding to cholesterol homeostasis. Our work demonstrates the potential for chemoproteomic fingerprints to translate between the chemical structure of signaling metabolites and their biological functions.

Results

Design and synthesis of probe 2

Chemoproteomics strategies for identifying cellular OHC interactions face a unique combination of challenges. In live cells, subtle features of chemical structure dictate the membrane environments that OHCs traverse and the targets to which they are exposed.^1,6 To capture the full complement of OHC interactions and the “fingerprint” of their cellular targets, chemoproteomics probes for cellular OHCs must preserve native membrane properties, retain oxygenation patterns recognized by specific receptors, and incorporate functionality to capture and identify live-cell targets. To address these requirements, we sought to modify natural OHC structures with a diazirine for live-cell photocrosslinking and an alkyne for downstream analysis. The diazirene functional group is benign to live cells, inert under ambient light, and can be photoactivated at 368 nm (long UV) to generate a reactive carbene that covalently crosslinks protein residues within a single-digit angstrom radius.⁹Diazirines in general are favored for their small size, bio-orthogonality, and low background labeling; for OHC probes in particular, this non-polar functional group can be introduced at a variety of positions without significantly influencing OHC membrane behavior. Alkynes likewise provide sterically minimal, biorthogonal modifications for “click”-based appendage of crosslinked proteins to virtually any affinity tag (e.g. biotin), fluorescent reporter, or desired functionality. The complementary reactivities of diazirenes and alkynes underlie their emerging popularity as chemoproteomics handles and are especially suited to OHC probes.

We first chose to fashion a bifunctional probe for the OHC metabolite 20(S)-OHC (1). This molecule has been detected in the brain and placenta,^10,11 and functions as a potent osteogenitor¹² and teratogen.¹³In addition to binding other proteins, 20(S)-OHC is a stereospecific ligand for the 7-transmembrane receptor Smoothened (Smo), a gatekeeper in developmental Hedgehog signaling.^7,8 Notably, the 20(R) epimer of this molecule (20(R)-hydroxycholesterol, 20(R)-OHC) is inactive at Smo. Stimulation of endogenous Smo activity by 20(S)-OHC and congeners can be assayed in murine embryonic fibroblasts, providing a platform for us to evaluate the biological activity of a 20(S)-OHC probe relative to the parent molecule.

To enable an unbiased analysis of 20(S)-OHC targets in cells, we positioned the alkyne and diazirine handles to preserve the C20(S)-OHC pharmacophore, the α- and β-face topologies of the tetracyclic ring system, the accessible C3-hydroxyl group, and the length and hydrophobicity of the C17 side chain (Fig. 1a). Synthetically, we envisioned the use of an intramolecular C-H activation for conversion of the C19 methyl group to an alkyne¹⁴ and a stereoselective Grignard addition to introduce a diazirine side chain to a C17 methyl ketone.¹⁵ In the forward sense, stereoselective hydrobromination of pregnenolone acetate (3) and photoinduced C-H activation of the C19 methyl group in 4¹⁴ provided ether 5 in 76% yield over two steps (Fig. 1b). Elaboration of the C19 primary alcohol to an alkyne via reductive elimination by elemental zinc in acetic acid, pyridinium chlorochromate oxidation to the C19 aldehyde 6, and homologation with the Seyferth-Glibert reagent provided the key intermediate 7. Gratifyingly, Grignard reaction to add the side chain at C20 afforded exclusively the 20(S) alcohol, whereupon liberation of the ketone in 8 and one-pot introduction of the C25 diazirine produced bifunctional photoaffinity probe 2 as an air- and ambient light-stable solid. In addition to furnishing 2, alkyne intermediate 7 represents a precursor to a library of bifunctional probes for side-chain oxysterols.

The structure of 2 was unambiguously established by 1D and 2D NMR spectroscopy. The presence of the diazirine was confirmed by a characteristic infrared N=N stretching frequency at 1644 cm⁻¹ and ultraviolet absorption at 353 nm. Irradiation of 2 in organic solvent at 368 nm resulted in progressive extrusion of N₂ with a half-life of 1.34 min (Fig. 1c). Thus, probe 2 differs from the parent structure by a count of only 4 atoms, ~1.5 Å in any dimension, and a logP of 0.48, and can be photoactivated to generate a reactive carbene for protein crosslinking.

To ensure that 2 preserves biological activity of 20(S)-OHC, we assessed its ability to activate Smo in Shh-LIGHT2 cells, a murine embryonic fibroblast line that bears a luminescent reporter of Smo-regulated Gli transcription (Fig. 1d). Commensurate with the known (S) stereoselectivity of 20-OHC:Smo binding, 20(S)-OHC induced reporter activity with an EC₅₀ of 8.4 μM, whereas 20(R)-OHC was inactive. Probe 2 induced Smo activity with an EC₅₀ of 1.2 μM, demonstrating that 2 retains activity at Smo. Here, the difference in response to 2 and 20(S)-OHC most likely reflects modest changes in solubility and/or membrane properties introduced by the probe functionalities, underscoring the sensitivity of certain OHC activities to subtle changes in structure. To assess biochemical engagement between 2 and Smo, we photocrosslinked 2 to YFP-Smo expressed in HEK293T cells and isolated Smo protein using a GFP/YFP nanobody (Fig. 1e). Click ligation of a biotin handle and visualization of crosslinked protein revealed that Smo, but not overexpressed GFP, was efficiently labeled by 2. These studies established that 2 retains activity at a stereospecific OHC receptor and can selectively crosslink target proteins in live cells, supporting its utility as a probe to map live-cell 20(S)-OHC interactions.

Establishment of OHC target labeling in live cells

To quantitatively catalogue the protein fingerprint interaction of 20(S)-OHC in live cells, we established conditions for live cell engagement, photochemical crosslinking, and biorthogonal tagging of target proteins (Fig. 2a). We performed these experiments in NIH-3T3 cells, which show an endogenous response to Smo activation and serve as a model to study osteogenesis and embryogenesis. For live cell engagement, we used an incubation time of 30 min to minimize effects of OHC-induced protein expression or degradation. To photocrosslink target proteins, we removed unbound probe and irradiated cells on ice at 368 nm for 5 min. To label crosslinked proteins, we lysed cells and performed a click reaction to ligate a TAMRA fluorophore to the alkyne handle in 2.

SDS-PAGE and in-gel fluorescence analysis of labeled proteins revealed a discrete set of bands that appeared in a probe-, UV-, and copper-dependent manner, indicating that they represented 2-crosslinked target proteins (Fig. 2b). Cell fractionation revealed that the majority of labeled proteins, including prominent bands at approximately 13 kDa, 21 kDa, and 37 kDa, appeared exclusively in the membrane fraction (Fig. 2c). To focus on these enriched targets and identify novel 20(S)-OHC interactors in the membrane proteome, we employed this fractionation method for remainder of our experiments. Analysis of the membrane proteome demonstrated these bands were labeled in a manner that showed good signal-to-background at 1 μM (Fig. 2d). Competition with unlabeled 20(S)-OHC revealed that the 21 kDa band was competable in a dose-dependent manner, showing 85% reduction in signal at a competitor concentration of 50 μM (Fig. 2e). The ability to label membrane proteins in a probe, UV, and click catalyst-dependent fashion in a manner that could be dose-dependently competed by 20(S)-OHC established the utility of 2 as a live cell chemoproteomics probe.

Analysis of enriched OHC targets

To profile the entire complement of 2 labeling in the membrane proteome, we replaced TAMRA azide in the click reaction with biotin azide to label crosslinked proteins. To isolate crosslinked proteins using streptavidin enrichment, we removed excess biotin through acetone precipitation, resolubilized the membrane proteome in SDS/Igepal buffer, and isolated biotinylated proteins on streptavidin agarose beads (Fig. 3a and Extended Data Fig. 1). For global target profiling, we used this protocol in the presence and absence of 1 μM 2 at a normalized concentration of 0.01% DMSO. On-bead digestion of streptavidin-enriched proteins and tandem isotope labeling of replicate samples enabled multiplexed real-time search-enabled MS³-based mass spectrometry analysis (RTS-SPS-MS³) of 2 interactors in the membrane proteome.

Our results revealed 12 proteins that were enriched by 25-fold relative to DMSO alone, and 106 proteins that were enriched by 5- to 25-fold at a p-value of < 0.002 (Fig. 3b). Comparison with transcript levels from RNA-seq analysis¹⁶ revealed no significant correlation between target protein enrichment and expression, indicating that enhancement was not solely a reflection of abundance (Extended Data Fig. 2c). The most enriched targets consisted of proteins previously established to bind OHCs (NPC1¹⁷ and Cav1¹⁸), cholesterol-binding proteins (Vdac1¹⁹), and proteins directly involved in cholesterol homeostasis (Tmem97²⁰). The set also included metabolic proteins that act on sterol substrates (Ldah,²¹ Ephx1²²) or participate in redox complexes associated with cholesterol biosynthesis (Vkorc1l1²³). Three of the proteins (Nptn and Bsg,²⁴Endod1²⁵) function as adaptors that control trafficking and activity of specialized membrane protein complexes. Performing our affinity enrichment protocol using label-free instead of TMT analysis revealed a nearly identical target profile with respect to identity and enrichment, demonstrating that the method of quantification did not significantly influence the target set (Extended Data Fig. 2b).

A key aspect of OHC function involves the unique effect of these molecules on membrane proteins sensitive to cholesterol content.^3,4,5,6 Accordingly, network analysis of OHC targets can reveal membrane topologies and cellular circuits that are responsive to low affinity and/or combinatorial OHC interactions. To identify ontologies associated with the 2 target fingerprint, we performed Gene Set Enrichment Analysis (GSEA) using 2-enriched proteins.²⁶ GSEA compares genetic and/or proteomic data to a database of curated gene sets, termed molecular signatures, that are associated with specific ontologies and diseases. The analysis then provides a rank-ordered list of molecular signatures that are overrepresented in the input data. Using the Gene Ontology (GO) gene set collection for GSEA with 2-enriched targets revealed a top list of 15 GO terms for cellular components, molecular functions, and biological processes with an FDR of < 0.1 (Fig. 3c). Among the most highly enriched membrane components were the Golgi apparatus and plasma membrane-ER networks, consistent with the observed localization of 20(S)-OHC to the Golgi and perinuclear regions,²⁷ and in contrast to the predominant localization of cholesterol to the plasma membrane.¹ Correspondingly, protein interactors of 2were associated with GO molecular functions that involved molecular signal transduction and transport across the membrane. The most highly represented GO biological processes included membrane lipid metabolism and sphingolipid metabolism in particular, in line with the observation that OHCs posttranslationally enhance the activity of enzymes involved in sphingomyelin biosynthesis.²⁸

To identify associations between 2-binding proteins and processes in human health and disease, we performed GSEA using the immunologic and oncogenic signature gene set collections (Fig. 3d,​,eeand Supplementary Table 1). The immunologic gene set with the highest representation of 2-enriched targets was GSE23505_IL6_IL1_VS_IL6_IL1_IL23_TREATED_CD4_TCELL_DN (NES = 2.92, FDR = 0.009). This gene set is downregulated in RORɣ-dependent, TGFβ-independent initiation of CD4+T cell differentiation into Th17 helper cells. The oncogenic gene set containing the most 2-enriched targets was TBK1.DF_UP (NES = 2.14, FDR = 0.071), a set of genes upregulated by TANK binding kinase 1 in KRAS-mutant lung adenocarcinoma cells. Since targeting of TBK1 in KRAS mutant cancer cells is synthetically lethal, inhibition of OHC-coordinated processes in these cells may represent an avenue for the treatment of refractory KRAS mutant cancers. The identification of OHC target proteins in immune system function and cancer signaling highlights new avenues of investigation for drug design and therapeutic targeting.

Identification of enriched and competable OHC targets

We next sought to determine which live-cell OHC interacting proteins could be out-competed by unlabeled 20(S)-OHC. To optimize the competability of 20(S)-OHC interactions, we focused on conditions that reduced non-specific labeling and enhanced sensitivity to competition. To do so, we pre-complexed competitor molecules with methyl-β-cyclodextrin (MβCD), a water-soluble cavitand that reversibly binds sterols and delivers them to cell membranes.²⁹ Co-application of 0.5 mM MβCD with 1 μM 2 in the presence or absence of 50 μM 20(S)-OHC competitor reproducibly solubilized all sterols and provided robust competition of the 21 kDa band identified by in-gel analysis (Fig. 2e). Because MβCD can perturb cellular cholesterol content, we identified an MβCD concentration that preserved equal intensities of non-competable bands in the presence and absence of competitor (Extended Data Fig. 3).

Our competition experiments revealed 12 proteins that were enriched by 2 by more than 7-fold at a p-value of 0.05 (Fig. 4a,​,b).b). Comparison of our enriched targets in the presence and absence of MβCD demonstrated that, while MβCD reduced the magnitude of fold changes versus DMSO, neither the identity nor the relative ranking of probe targets were significantly altered (Fig. 4b and Extended Data Fig. 3c). We again observed the proteins Tmem97, Ephx1, Cav1, Vkorc1l1, Ldah, Vdac2, and Bsg in the top 5% of target enrichment, comprising ~60% of the most enriched proteins in our previous experiments (Fig. 3b). Another 89 targets were enriched at between 2.75 and 7-fold at a p-value of 0.05, representing a selection of transmembrane proteins, signal transduction proteins, lipid biosynthetic enzymes, and membrane trafficking/adaptor proteins identified in our previous GO analysis. Enriched proteins in these experiments also involved in Golgi membrane homeostasis (Tmem199³⁰), transmembrane proteins of unannotated function (Tmem238, Smim4), and the caveolin isoform Cav2.

As anticipated, only a subset of 2 interactions were competable by 20(S)-OHC (Fig. 4d,​,e).e). In this dataset, a sharp divide separated the top 0.3% of proteins from less competable interactors. The most competable proteins encompassed the highly enriched targets Tmem97, Ephx1, and Cav1. In addition, the list included Abcb1 (P-glycoprotein 1), an ATP-dependent multidrug/steroid transporter,³¹and Arxes1/2, a retrotransposed protease gene on the X-chromosome that plays a role in adipogenesis.³²Both Abcb1b and Arxes1/2 also appear with in the top 2% of proteins enriched by 2.

The full fingerprint of direct OHC binding proteins provided us with an opportunity to probe the engagement of OHCs and proteins with relevant biological functions. Accordingly, we selected a set of functionally relevant 20(S)-OHC targets and validated their engagement with 2 by live-cell crosslinking, biotin-streptavidin enrichment, and Western blot detection using specific antibodies (Fig. 4f). A highly competable protein identified in our data, Gas1, is an upstream Smo regulator that coordinates the Hedgehog signaling pathway in embryonic development. Competable binding of Gas1 by 20(S)-OHC complements recent findings that Gas1 localizes to cholesterol microdomains as an obligate part of its Hh signaling activity,³³ and suggests that endogenous OHCs may directly or indirectly contribute to this mechanism of regulation. The moderately competable protein isoforms Vdac 1 and 2 are known to control steroidogenesis by directing the transport of cholesterol from the outer to the inner mitochondrial membrane for conversion to pregnenolone by Cyp11a1,³⁴ suggesting a possible regulatory role for OHCs. Potentially functional but less competable OHC binding was also observed with the scaffolding protein Reep5, which plays a structural role in the architecture of sarcolemmal membranes in muscle cells,³⁵ the protein Gpr107, which chaperones receptors to organelle membranes,³⁶ and the adaptor protein Tmem199, an assembly factor in vacuolar ATPase complexes that control energy-dependent acidification of cellular organelles.³⁰ In each of these cases, 2 captured endogenous protein to an extent that correlated with MS-based abundance, verifying that our proteome-wide profiling can provide a quantitative catalogue of functional 2-interacting proteins in living cells (Extended Data Fig. 4).

Discovery of Tmem97 as a selective OHC target

The most enriched and competable protein identified in our studies was Tmem97, a transmembrane protein with a molecular weight of 21 kDa– approximately the same molecular weight as the competable band identified in our in gel fluorescence analysis (Fig. 2e). Tmem97, also known as the σ2 receptor, is a protein biomarker that is overexpressed in proliferative tumors and a target for PET imaging in the clinic.³⁷ Originally characterized by its pharmacological profile, Tmem97 has also been investigated as a non-opioid target for psychiatric conditions and neuropathic pain.²⁰ Identification of the Tmem97 gene in 2017 and genetic perturbation studies subsequently established an integral role for Tmem97 in cholesterol homeostasis, where it regulates levels of the cholesterol transporter protein NPC1.^38,39,40 For its utility as a cancer radioimaging agent and its implication in lysosomal cholesterol storage diseases, Tmem97 is a valuable therapeutic target. However, no endogenous ligand for Tmem97 has been established.

To determine whether Tmem97 contributed to the 21 kDa band identified in our gel-based assays, we took advantage of ligands selective for Tmem97 or the structurally unrelated σ1 receptor. Using the σ1 igand PRE-084 or the Tmem97 ligand BIMU-8 as competitors, live-cell crosslinking to 2, TAMRA labeling, and SDS-PAGE analysis revealed that the Tmem97 ligand, but not the σ1 receptor ligand, effectively out-competed labeling by 2 (Fig. 5a). Biotin labeling, streptavidin pulldown, and immunodetection using a Tmem97 antibody likewise confirmed that Tmem97 was recovered only in the presence of the σ1 receptor ligand, supporting the identification of Tmem97 as a 2-labeled target.

To confirm that Tmem97 was the major component of the 2-labeled 21 kDa band, we used CRISPR/Cas9 to ablate expression of Tmem97 in a population of NIH-3T3 cells (Fig. 5b). Photoaffinity crosslinking of 2 in wild-type and Tmem97-knockout cells demonstrated that loss of Tmem97 eliminated the prominent TAMRA-labeled band at 21 kDa. To evaluate specific competition of Tmem97, we crosslinked 2 to cells in the presence of varying amounts of 20(S)-OHC, enriched crosslinked proteins, and examined the intensity of Tmem97 immunodetection by Western blot (Extended Data Fig. 5a). Increasing concentrations of 20(S)-OHC dose-dependently reduced the strength of the Tmem97 band in a manner proportional to loss of the probe intensity at 21 kDa. To assess whether Tmeme97 labeling was selective for 2, we crosslinked cellular proteins using the commercially available cholesterol probe PhotoClick Cholesterol (14) (Extended Data Fig. 5b). Under identical conditions, 2, but not PhotoClick Cholesterol,⁴¹competably labeled overexpressed Tmem97 in HEK293T cells. Together, these experiments indicate that Tmem97 is a dose-dependently competable, selective target of 2.

Next we sought to determine ability of structurally distinct small molecules and cholesterol metabolites to out-compete labeling by 2. Neither the long-chain lipid palmitoylethanolamide (PEA, 9) nor the polyhydroxylated carbocycle inositol (10) could effectively reduce labeling by 2, indicating that neither bound to Tmem97 at the same site as 2 and 20(S)-OHC (Fig. 5c and Extended Data Fig. 5c). To determine whether Tmem97 could distinguish hydroxylation patterns within the sterol structure, we evaluated competition by unmodified cholesterol (11) or the regioisomeric oxysterol 25-OHC (12). While cholesterol itself was a poor competitor of 2, 25-OHC could partially outcompete labeling by 2, indicating that Tmem97 is sensitive to the extent and pattern of sterol hydroxylation. Finally, we tested whether the configuration of the C20 alcohol could differentiate competition by 20(S)- and 20(R)-OHC (13). Notably, the 20(S) stereochemistry is produced endogenously by Cyp11a1 oxidation of cholesterol en route to pregnenolone.⁴² Competition with either the C20(S) or the C20(R) epimer revealed that 20(R)-OHC competed only 50% of probe labeling at a concentration of 50 μM, whereas competition by 50 μM 20(S)-OHC completely abolished labeling, indicating a stereochemical preference for the C20(S)-OH interaction.

Tmem97 is phylogenetically related to the enzyme 3-β-hydroxysteroid-Δ⁸,Δ⁷-isomerase (Emopamil-binding protein, EBP), which catalyzes migration of the C8-C9 double bond in zymosterol/zymostenol to its C7-C8 position in lathosterol/dehydrolathosterol.⁴³ Despite their evolutionary relationship, Tmem97 lacks an acidic residue homologous to E122 in EBP (human numbering) and has no discernable enzymatic activity.²⁰ To determine a binding site for 20(S)-OHC within Tmem97, we used the crystal structure of human EBP (PDB 6OHT)⁴⁴ as a template to predict the structure of mouse Tmem97 (82% pairwise identity with human Tmem97) using the Robetta server.⁴⁵ We embedded the resulting model within a POPC membrane and performed all-atom molecular dynamics simulations to equilibrate the membrane-bound Tmem97 structure. Docking of 20(S)-OHC into this model produced a stable pose in which 20(S)-OHC binds to several residues homologous to those in the active site of EBP (Extended Data Fig. 6a). Convincingly, while this manuscript was in revision, the first crystal structure of Tmem97 in complex with synthetic σ2 ligands was reported, evidencing a ligand binding site with striking resemblance to our homology-based model.⁴⁶ As in EBP and other sterol-binding proteins, an array of hydrophobic and aromatic residues line a central Tmem97 ligand-binding cavity with a helical “cap” at the top. In our docked 20(S)-OHC:Tmem97 model, the C3 hydroxyl group of 20(S)-OHC forms a hydrogen bond to the imidazole side chain of H106, while the iso-octyl tail is accommodated by a hydrophobic pocket that includes F66, P113, V146, Y147, and Y150. Hydrophobic interactions between the iso-octyl side chain and these residues would disfavor introduction of a polar group at C25, providing a possible rationale for reduced competition of 2 by 25-OHC versus 20(S)-OHC.

To ascertain whether residues in the Tmem97 binding pocket influenced the extent of 2 labeling or degree of competition by 20(S)-OHC, we transiently overexpressed epitope-tagged Tmem97 and constructs containing mutations at residues near 20(S)-OHC in HEK293T cells (Fig. 5d). While the F88A mutant protein was not expressed, the Y150A, E61A, and W95A mutants were robustly produced, appearing ~3 kDa higher in apparent MW than endogenous Tmem97 due to the presence of a FLAG-Myc epitope tag. Interestingly, while the E61A and W95A mutants were labeled by 2 and competable by 20(S)-OHC to the same extent as the wild-type protein, the Y150A mutant was not labeled. Because Y150 is the closest polar residue to the C20-OHC alcohol in our docked model, we hypothesized that it might engage in polar and/or hydrogen bonding interactions via its phenolic hydroxyl group. To determine whether the Y150 side chain affected either the efficiency or structure-selectivity of Tmem97 labeling, we generated two additional Tmem97 mutants: Y150F, which retains an aromatic group but eliminates hydrogen bonding, and Y150S, which lacks aromaticity but retains the ability to form hydrogen bonds. Crosslinking and in-gel fluorescence analysis demonstrated that, while the Y150F mutant was labeled to approximately 60% of the wild-type protein, the Y150S showed no significant labeling (Fig. 5e and Extended Data Fig. 6b). Intriguingly, the Y150F mutation not only reduced competition by 20(S)-OHC, but eliminated stereoselective displacement by 20(S)-OHC over 20(R)-OHC. Together, these results suggest that the aromatic moiety of Y150 contributes to OHC binding affinity, while the phenolic hydroxyl group may confer a preference for engagement with the 20(S)-OHC epimer. These experiments identify discrete structural features of the Tmem97 binding site that dictate OHC:Tmem97 binding in cells, offering unique insight into the molecular details of their engagement.

20(S)-OHC enhances association of Tmem97 and NPC1

In 2009, Tmem97 was identified as a novel regulator of cholesterol homeostasis in a screen for proteins that influence cellular cholesterol content.³⁹ This and a subsequent study established that genetic knockdown of Tmem97 inhibits internalization of low-density lipoprotein particles and reduces cholesterol concentrations in lysosomes.⁴⁰ These effects coincide with an increase in the levels of NPC1 protein- a central coordinator of cholesterol distribution that exports free cholesterol from lysosomes. Tmem97 and NPC1 co-immunoprecipitate and function in a manner that relies the Tmem97 ER-targeting motif, consistent with a role for Tmem97 as an ER-localized NPC1 chaperone protein.

Localized modulation of protein-protein interactions is a hallmark of oxysterol metabolite signaling.^3,4,5,6In the most well-established of these mechanisms, selective ligation of INSIG by 25-OHC induces its association with the ER-localized sensor SCAP, activating expression of cholesterol biosynthetic genes.⁴⁷ OHC-mediated protein-protein interactions also regulate the degradation of HMG-CoA reductase,⁴⁸ the affinity of adrenodoxin-CYP11 complexes in pregnenolone synthesis,⁴⁹ tumor-associated dimerization of surface receptors,⁵⁰ and other processes. OHC regulation of protein activity extends to soluble and membrane-associated proteins, including members of the OSBP/OSBPL, StAR/StAr-related lipid transfer domain-containing, and Aster protein families, and nuclear hormone receptors such as LXRs, RORɣ, and ERα/β.^4,5

Based on the pervasive examples of oxysterol-regulated protein-protein interactions, we hypothesized that ligation of Tmem97 by 20(S)-OHC may influence its association with NPC1. To test whether binding of 20(S)-OHC to Tmem97 affects its physical interaction with NPC1, we examined co-immunoprecipitation of Tmem97 and NPC1 in cells treated with varying concentrations of 20(S)-OHC. To avoid complications associated with OHC-induced upregulation of Tmem97 via SREBP,³⁹ we overexpressed epitope-tagged Tmem97 and performed our experiments in HEK293T cells, which express robust levels of endogenous NPC1. Incubation of Tmem97-expressing cells with 20(S)-OHC followed by capture of NPC1 using Tmem97 as bait evidenced a robust increase in Tmem97:NPC1 association at 10 μM (Fig. 6a). Likewise, incubation of Tmem97-expressing cells with 10 μM 20(S)-OHC, and capture of Tmem97 using endogenous NPC1 as bait showed a significant enhancement of the Tmem97:NPC1 interaction (Fig. 6b). Importantly, whole-cell Tmem97:NPC1 ratios were unaffected by 20(S)-OHC, demonstrating that 20(S)-OHC selectively impacts the Tmem97:NPC1 interaction (Fig. 6a,​,b).b). As observed previously, overexpression of Tmem97 had no effect on overall cholesterol distributions, as determined by filipin staining,⁴⁰ in either the absence or presence of 20(S)-OHC. The observation that 20(S)-OHC selectively influences Tmem97:NPC1 association without changing bulk cellular properties is consistent with a localized interaction between Tmem97 and NPC1.

Compellingly, other reports have demonstrated that OHCs act as chaperones for NPC1, although the exact mechanism of the OHC:NPC1 interaction remains undetermined.¹⁷ Our results raise the intriguing possibility this chaperoning activity may in part be mediated by a ternary interaction between OHCs, Tmem97, and NPC1 in a mechanism reminiscent of the 25-OHC:INSIG:SCAP interaction. We note that our studies are not without limitations: overexpressed Tmem97 may not fully recapitulate the behavior of endogenous Tmem97, and the relatively high 20(S)-OHC concentrations required to effect Tmem97:NPC1 perturbations might arise from the difference between applied and subcellular OHC concentrations. To more precisely examine local phenomena, we are currently extending our use of probe 2 as an imaging agent to visualize cellular interactions between 20(S)-OHC, Tmem97, and NPC1. Further studies to assess localized cholesterol concentrations, 20(S)-OHC-regulated trafficking and/or modification of NPC1, and structural features of the Tmem97:NPC1 interaction in cholesterol regulation are underway.

Discussion

While cholesterol is the most abundant lipid present in mammalian cell membranes, OHCs are transient species that exist at low concentrations in cells. Additional hydroxylation of OHCs relative to cholesterol confers these molecules with privileged access to cholesterol binding sites and membrane environments, where their presence can profoundly disrupt locales and resident proteins. The activity of OHCs in cells is a function of their affinity for 

specific receptors and their constellation of cellular targets. Well-known for their signaling roles, the complete set of OHC binding remain virtually unknown, and therapeutic opportunities associated with their direct targets and functions are largely untapped.

In this work, we describe our design, synthesis, and application of a chemoproteomics OHC probe to fingerprint OHC interactions in live mammalian cells. By incorporating alkyne and diazirine functional groups within the signaling molecule 20(S)-OHC, we establish a probe that precisely mimics the natural molecule and preserves its signaling activity in cells. We create a reproducible, quantitative protocol for competing targets of 2 in the native environments and perform multiplexed, proteome-wide analysis. We reveal OHC targets on a spectrum of competability that have functional roles in signal transduction, protein trafficking, and membrane lipid metabolism, and we identify specific processes in immune response and cancer that are functionally integrated with OHC target proteins. We demonstrate that the orphan receptor Tmem97 (also known as the σ2 receptor) is a stereo- and regioselective target of OHCs in live cells, evidencing its functional roles in NPC1-mediated cholesterol metabolism and providing a new template to design molecules that intervene in its functions.

We note that the membrane proteome examined in this study excludes nuclear hormone receptors that bind OHCs; we are currently profiling the nuclear fraction to quantify OHC engagement with known targets and yet-unidentified OHC receptors. In the future, chemoproteomics analysis of sterol and other hydrophobic metabolites will benefit from new reagents for small molecule delivery to cells, such as synthetic LDL particles and next-generation cavitands. Improved methods for membrane protein solubilization, combined with powerful advances in liquid chromatography/mass spectrometry of hydrophobic peptides, can likewise enhance the resolution and detection of target proteins. Critically, enhanced coverage hinges on fundamental improvements in diazirine photocrosslinking chemistry and continued optimization of click labeling protocols. In synergy with appropriate data analysis, this multiplexed chemoproteomic profiling provides a powerful platform to inform target prediction and aid the design of metabolite-inspired drugs. More broadly, the identification of metabolite targets in cells integrates chemical structure and biological activity, offering new dimensions to our understanding of human health.