Sterol Binding Research Articles

Structural and biochemical analysis of ligand binding in yeast Niemann-Pick type C1-related protein.

In eukaryotes, integration of sterols into the vacuolar/lysosomal membrane is critically dependent on the Niemann-Pick type C (NPC) system. The system consists of an integral membrane protein, called NCR1 in yeast, and NPC2, a luminal soluble protein that transfers sterols to the N-terminal domain (NTD) of NCR1 before membrane integration. Both proteins have been implicated in sterol homeostasis of yeast and humans. Here, we investigate sterol and lipid binding of the NCR1/NPC2 transport system and determine crystal structures of the sterol binding NTD. The NTD binds both ergosterol and cholesterol, with nearly identical conformations of the binding pocket. Apart from sterols, the NTD can also bind fluorescent analogs of phosphatidylinositol, phosphatidylcholine, and phosphatidylserine, as well as sphingosine and ceramide. We confirm the multi-lipid scope of the NCR1/NPC2 system using photo-crosslinkable and clickable lipid analogs, namely, pac-cholesterol, pac-sphingosine, and pac-ceramide. Finally, we reconstitute the transfer of pac-sphingosine from NPC2 to the NTD in vitro. Collectively, our results support that the yeast NPC system can work as versatile machinery for vacuolar homeostasis of structurally diverse lipids, besides ergosterol.

Sterolysin from a 1950s culture of Karlodinium veneficum (aka Gymnodinium veneficum Ballantine) forms lethal sterol dependent membrane pores

In 1957 Abbott and Ballantine described a highly toxic activity from a dinoflagellate isolated from the English Channel in 1949 by Mary Park. From a culture maintained at Plymouth Laboratory since 1950, we have been able to isolate two toxic molecules (abbotoxin and 59-E-Chloro-abbotoxin), determine the planar structures by analysis of HRMS and 1D and 2D NMR spectra, and found them to be karlotoxin (KmTx) congeners. Both toxins kill larval zebrafish with symptoms identical to those described by Abbot and Ballantine for gobies (Gobius virescens). Using surface plasma resonance the sterol binding specificity of karlotoxins is shown to require desmethyl sterols. Our results with black lipid membranes indicate that karlotoxin forms large-conductance channels in the lipid membrane, which are characterized by large ionic conductance, poor ionic selectivity, and a complex gating behavior that exhibits strong voltage dependence and multiple gating patterns. In addition, we show that KmTx 2 pore formation is a highly targeted mechanism involving sterol-specificity. This is the first report of the functional properties of the membrane pores formed by karlotoxins and is consistent with the initial observations of Abbott and Ballantine from 1957.

ORMDL mislocalization by impaired autophagy in Niemann-Pick type C disease leads to increased de novo sphingolipid biosynthesis

Niemann-Pick type C1 (NPC1) disease is a rare neurodegenerative cholesterol and sphingolipid storage disorder primarily due to mutations in the cholesterol-trafficking protein NPC1. In addition to catabolic-derived sphingolipids, NPC1 dysfunction also leads to an increase in de novo sphingolipid biosynthesis, yet little is known about the cellular mechanism involved. Although deletion of NPC1 or inhibition of the NPC1 sterol binding domain enhanced de novo sphingolipid biosynthesis, surprisingly levels of the ORMDLs, the regulatory subunits of serine palmitoyltransferase (SPT), the rate-limiting step in sphingolipid biosynthesis, were also greatly increased. Nevertheless, less ORMDL was bound in the SPT-ORMDL complex despite elevated ceramide levels. Instead, ORMDL colocalized with p62, the selective autophagy receptor, and accumulated in stalled autophagosomes due to defective autophagy in NPC1 disease cells. Restoration of autophagic flux with N-acetyl-L-leucine in NPC1 deleted cells decreased ORMDL accumulation in autophagosomes and reduced de novo sphingolipid biosynthesis and their accumulation. This study revealed a previously unknown link between de novo sphingolipid biosynthesis, ORMDL, and autophagic defects present in NCP1 disease. In addition, we provide further evidence and mechanistic insight for the beneficial role of N-acetyl-L-leucine treatment for NPC1 disease which is presently awaiting approval from the Food and Drug Administration and the European Medicines Agency.

Investigation of the insecticidal potential of curcumin derivatives that target the Helicoverpa armigera sterol carrier protein-2

Cotton bollworm (Helicoverpa armigera) is a highly polyphagous, widely prevalent, and persistent Old World insect pest that affects numerous important crops that are directly consumed by people, including tomato, cotton, pigeon pea, chickpea, rice, sorghum, and cowpea. Insects do not synthesize steroids but obtain them from their diet. Inhibition of dietary uptake of steroids by insects is a potentially effective insecticidal mechanism that should not be toxic to humans and other mammals, who synthesize their steroids. Ten curcumin derivatives were tested against H. armigera sterol carrier protein-2 (HaSCP-2) for their potential as insecticidal agents. Curcumin derivatives were initially docked at the binding site of HaSCP-2 to determine their binding affinities and plausible binding modes. The binding modes predominantly show hydrophobic interactions of derivatives with Phe53, Phe110, and Phe89 as core interacting residues in the active site. Validation of in silico results was carried out by performing a fluorescence binding and displacement assay to determine the binding affinities of curcumin derivatives. Among a collection of curcumin derivatives tested, Cur10 showed the lowest IC50 value of 9.64 μM, while Cur07 was 19.86 μM, and Cur06 was 20.79 μM. There was a significant negative correlation between the ability of the curcumin derivatives tested to displace the fluorescent probe from the sterol binding site of HaSCP-2 and to inhibit Sf9 insect cell growth in culture, which is consistent with the curcumin derivatives acting by the novel mechanism of blocking sterol uptake. Then molecular dynamics simulation studies validated the predicted binding modes and the interactions of curcumin derivatives with HaSCP-2 protein. In conclusion, these studies support the potential use of curcumin derivatives as insecticidal agents.

Novel sterol binding domains in bacteria.

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.

Novel sterol binding domains in bacteria

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.

Direct binding to sterols accelerates endoplasmic reticulum-associated degradation of HMG CoA reductase

The maintenance of cholesterol homeostasis is crucial for normal function at both the cellular and organismal levels. Two integral membrane proteins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and Scap, are key targets of a complex feedback regulatory system that operates to ensure cholesterol homeostasis. HMGCR catalyzes the rate-limiting step in the transformation of the 2-carbon precursor acetate to 27-carbon cholesterol. Scap mediates proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a membrane-bound transcription factor that controls expression of genes involved in the synthesis and uptake of cholesterol. Sterol accumulation triggers binding of HMGCR to endoplasmic reticulum (ER)-localized Insig proteins, leading to the enzyme's ubiquitination and proteasome-mediated ER-associated degradation (ERAD). Sterols also induce binding of Insigs to Scap, which leads to sequestration of Scap and its bound SREBP-2 in the ER, thereby preventing proteolytic activation of SREBP-2 in the Golgi. The oxygenated cholesterol derivative 25-hydroxycholesterol (25HC) and the methylated cholesterol synthesis intermediate 24,25-dihydrolanosterol (DHL) differentially modulate HMGCR and Scap. While both sterols promote binding of HMGCR to Insigs for ubiquitination and subsequent ERAD, only 25HC inhibits the Scap-mediated proteolytic activation of SREBP-2. We showed previously that 1,1-bisphosphonate esters mimic DHL, accelerating ERAD of HMGCR while sparing SREBP-2 activation. Building on these results, our current studies reveal specific, Insig-independent photoaffinity labeling of HMGCR by photoactivatable derivatives of the 1,1-bisphosphonate ester SRP-3042 and 25HC. These findings disclose a direct sterol binding mechanism as the trigger that initiates the HMGCR ERAD pathway, providing valuable insights into the intricate mechanisms that govern cholesterol homeostasis.

Sterol interactions influence the function of Wsc sensors

The Wsc1, Wsc2, and Wsc3 proteins are essential cell surface sensors that respond to cell wall perturbation by activating the cell wall integrity pathway (CWIP). We show here that in situ production of cholesterol (in place of ergosterol) induces hyper-phosphorylation of Slt2, the MAPK of the CWIP, and upregulates cell wall biosynthesis. Deletion of all three Wsc genes in K. phaffii reverts these phenotypes. In the cholesterol-producing strain, both Wsc1 and Wsc3 accumulate in the plasma membrane. Close inspection of the transmembrane domains of all three Wsc proteins predicted by AlphaFold2 revealed the presence of CRAC sterol-binding motifs. Experiments using a photoreactive cholesterol derivative indicate intimate interaction of this sterol with the Wsc transmembrane domain, and this apparent sterol binding was abrogated in Wsc mutants with substitutions in the CRAC motif. We also observed cholesterol interaction with CRAC-like motifs in the transmembrane domains of mammalian integrins, analogs of Wsc proteins. Our results suggest that proper signaling of the Wsc sensors requires highly specific binding of the native endogenous terminal sterol, ergosterol.

Substrate binding and inhibition of the anion exchanger 1 transporter.

Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.

Mutation on NPC1L1 NTD and the sterol binding stability: molecular dynamics study

Mutation on NPC1L1 NTD and the sterol binding stability: molecular dynamics study

Identification of key upregulated genes involved in foam cell formation and the modulatory role of statin therapy

We aimed to investigate the possible effect of statins on important genes/proteins involved in foam cell formation. The gene expression profile of the GSE9874, GSE54666, and GSE7138from the Omnibus database were usedto identify genes involved in foam cell formation. The protein-protein interaction (PPI) network and MCODE analysis of the intersection of three databases were analyzed. We used molecular docking analysis to investigate the possible interaction of different statins with the overexpressed hub genes obtained from PPI analysis. The intersection among the three datasets showed 54 upregulated and 26 down-regulated genes. The most critical overexpressed genes/proteins obtained as hub genes included: G6PD, NPC1, ABCA1, ABCG1, PGD, PLIN2, PPAP2B, and TXNRD1 based on PPI analysis. Functional enrichment analysis of 81 intersection DEGs at the biological process level focusing on the cholesterol metabolic process, secondary alcohol biosynthetic process and the cholesterol biosynthetic process. Under cellular components, the analysis confirmed that these 81 intersection DEGs were mainly applied in endoplasmic reticulum membrane, lysosome and lytic vacuole. The molecular functions were identified as sterol binding, oxidoreductase activity and NADP binding. The molecular docking showed that all statins appear to affect important protein targets overexpressed in foam cell formation. However, lipophilic statins, especially pitavastatin and lovastatin, had a greater effect than hydrophilic statins. The most significant protein target of all the overexpressed genes interacting with all statin types was ABCA1. The effect of lipophilic statins was shown for several critical proteins in foam cell formation.

Mechanism of the cholesterol transport in smoothened.

Cholesterol (CHL) has been identified as the endogenous agonist of Smoothened (SMO), a Class F G-Protein Coupled Receptor (GPCR). SMO, a member of the Hedgehog signaling pathway, is endogenously inhibited by Patched (PTCH). How PTCH inhibits SMO is unclear - however, the leading hypothesis states that PTCH controls SMO's accessibility to membrane cholesterol by being a putative cholesterol transporter. On PTCH's inhibition by Sonic Hedgehog (ShH), cholesterol can then travel through the upper leaflet in SMO to the sterol binding site in SMO CRD. SMO structures with sterols at different points along the pathway have been observed, but a study connecting the different sterol poses is lacking. Here, we use millisecond-scale atomistic molecular dynamics simulations in tandem with Markov State Modeling (MSM) to probe the cholesterol transport dynamics of SMO. We observe that SMO can indeed transport cholesterol from the membrane to the CRD site, a probable path exists for the same. A maximum barrier of ∼7.5 kcal/mol is observed at the membrane-SMO interface for the entry of cholesterol into the SMO Transmembrane Domain (TMD). This entry involves the rocking motion of cholesterol, which is elucidated by the tail-first entry of cholesterol entry in the TMD. This is followed by translation to the TMD-CRD interface, where CHL might showing flipping motions. The final step is the translation from the TMD-CRD interface to the CRD binding site. We observe that simulations with cholesterol make SMO inherently active, further corroborating the presence of multiple-binding sites of CHL in SMO. Thus, our study gives dynamical insights into a long-standing question in the field of endogenous SMO activation.

Measuring the Interaction of Sterols and Steroids with Proteins by Microscale Thermophoresis.

Determining the affinity and specificity of protein-lipid interactions is crucial for understanding the physiological function and mode of action of signaling lipids, including steroids. Here we describe a method that relies on microscale thermophoresis (MST) to monitor the binding of sterols and steroids to proteins. The protein of interest is expressed as a polyhistidine-tagged fusion in E. coli and purified by affinity chromatography on a nickel-based resin. The purified protein is then labeled with a fluorescent dye and incubated with a serial dilution of the lipid ligand. The protein-ligand interaction is monitored by MST, which detects the fraction of the protein bound to the ligand based on its altered mobility in a thermal gradient. A binding curve fitted to the measured data points is then used to calculate the corresponding dissociation constant.

Snapshots of ABCG1 and ABCG5/G8: A Sterol's Journey to Cross the Cellular Membranes.

The subfamily-G ATP-binding cassette (ABCG) transporters play important roles in regulating cholesterol homeostasis. Recent progress in the structural data of ABCG1 and ABCG5/G8 disclose putative sterol binding sites that suggest the possible cholesterol translocation pathway. ABCG1 and ABCG5/G8 share high similarity in the overall molecular architecture, and both transporters appear to use several unique structural motifs to facilitate cholesterol transport along this pathway, including the phenylalanine highway and the hydrophobic valve. Interestingly, ABCG5/G8 is known to transport cholesterol and phytosterols, whereas ABCG1 seems to exclusively transport cholesterol. Ligand docking analysis indeed suggests a difference in recruiting sterol molecules to the known sterol-binding sites. Here, we further discuss how the different and shared structural features are relevant to their physiological functions, and finally provide our perspective on future studies in ABCG cholesterol transporters.

Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain.

Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.

A Plasma Cuprome Exists With Predictors of Copper Status in Nepalese Children

ObjectivesCopper (Cu) is an essential micronutrient but, due to difficulties of measuring Cu by atomic absorption spectroscopy (AAS), plasma Cu status is rarely assessed in low income countries. A less costly assay is needed. We have explored whether plasma Cu concentration can be predicted by modelling strongly associated plasma proteins, assessed by mass spectrometry (MS), that could be assayed with other nutriproteomic biomarkers on one platform in the future.MethodsIn plasma samples from 500 Nepalese children 6–8 y of age, the mean (SD) plasma Cu concentration measured by AAS was 23.3 ± 5.7 mmol/L, with 13.6% classified as deficient (< 10 mmol/L). We quantified relative abundance of 982 plasma proteins by iTRAQ tandem MS following affinity depletion of six high abundance proteins (Cole J Nutr 2013) and correlated their relative abundance with plasma Cu by linear mixed effects regression. We defined a stringent plasma cuprome comprising proteins associated with Cu at a false discovery rate (q) < 0.01. Missing values were imputed and proteins were fit by forward, stepwise regression analysis, meeting an AIC reduction of ≥ 30, to model plasma Cu status.ResultsThere were 134 (q < 0.01) proteins in the plasma cuprome. Among 62 positive correlates were ceruloplasm (r = 0.65), 9 complement proteins (r = 0.29 to 0.45), 4 serpin protease inhibitors (r = 0.31 to 0.43) with others involved in coagulation, 5 LRR structural motifs as found in toll-like receptors (r = 0.35 to 0.58), signaling enzymes such as CDC42BPA (r = 0.70) and proinflammatory reactants and regulators such as CRP, AGP, amyloids and TNIP1 (r = 0.40 to 0.56). Among 72 negative correlates (r = −0.29 to −0.42) are lipid and sterol binding and transport proteins including apolipoproteins/lipocalins, enzyme regulating proteins, extracellular adhesion, signaling and matrix proteins. Different members of serine peptidase inhibitor members were among + and – correlates. Forward stepwise regression fit 6 proteins (CP, CDC42BPA, LRRC47, TDRD9, LLRIQ1, LRRCC1) that explain 76.5% of the variance (R2) in plasma Cu, sufficient for predicting plasma Cu status of a population.ConclusionsA large, diverse plasma cuprome exists, as revealed in young Nepalese children, from which six proteins may adequately predict plasma Cu status.Funding SourcesBill & Melinda Gates Foundation (OPPGH5241) & Johns Hopkins University.

Protein Kinase A Catalytic subunit:Smoothened interface ‐ Role of a parallel holoenzyme in negative feedback in Hedgehog Signaling

During signal transduction in the Hedgehog pathway, the G protein‐coupled receptor (GPCR) Smoothened (SMO) signals intracellularly via a novel mechanism involving sequestration of the Protein Kinase A (PKA) catalytic (C)‐subunit at the membrane. Activation of Hedgehog signaling leads to negative feedback regulation mediated through inhibition of PKA‐C by SMO at the ciliary membrane. While it is known that PKA‐C interacts with SMO at its C‐terminal proximal cytoplasmic tail domain (pCT), the dynamics of PKA‐C:SMO interactions is poorly understood. Here, we report a map of the PKA‐C:SMO interface by Amide Hydrogen Deuterium Exchange Mass spectrometry (HDXMS) over 1‐10 min timescales. SMO binding on PKA‐C resulted in global decreases in deuterium exchange (Dex). Peptides spanning the kinase activation loop (188‐198), P+1 loop (199‐211) and pseudo‐substrate recognition site between β5 and αD (129‐145) showed significant protection (&gt;0.5 Da) when bound to SMO, identifying these regions as the SMO binding interface on PKA‐C. Interestingly, the footprint of SMO binding on PKA‐C was identical to that of the RIa regulatory subunit in PKA‐I holoenzymes. Furthermore, increased Dex (0.4 Da) was observed at the region spanning αC and β4 (83‐100), indicating that SMO binding destabilizes the active kinase conformation to allosterically inhibit PKA‐C. Correspondingly on SMO, PKA‐C binding elicited only low magnitudes of Dex protection (0.4 Da) in regions flanking the pseudo substrate site (573‐590, 622‐631). Increased Dex (0.4 Da) was observed at the sterol binding pocket (96‐112) in the extracellular Cysteine rich domain, indicating that PKA‐C binding may allosterically impact SMO activation by modulating its ability to bind sterols. A significant increase in Dex (0.9 Da) was observed at SMO helix 8 (537‐546) which is positioned parallel to the membrane, indicating that C binding promoted a disordered helix 8 conformation. We speculate that the pCT exists in a membrane‐bound or folded conformation in free SMO, but PKA‐C interaction switches the binding mode of pCT leading to a significantly Dex‐protected state. Thus, PKA‐C:SMO interactions reveal an unexpected intersection between cyclic nucleotide signaling and the Hedgehog pathway, defining a novel mechanism of PKA‐C regulation beyond the canonical PKA signalosome.

Hedgehog signalling in bone and osteoarthritis: the role of Smoothened and cholesterol.

Hedgehog signalling is essential for development, crucial for normal anatomical arrangement and activated during tissue damage repair. Dysregulation of hedgehog signalling is associated with cancer, developmental disorders and other diseases including osteoarthritis (OA). The hedgehog gene was first discovered in Drosophila melanogaster, and the pathway is evolutionarily conserved in most animals. Although there are several hedgehog ligands with different protein expression patterns, they share a common plasma membrane receptor, Patched1 and hedgehog signalling pathway activation is transduced through the G-protein-coupled receptor-like protein Smoothened (SMO) and downstream effectors. Functional assays revealed that activation of SMO is dependent on sterol binding, and cholesterol was observed bound to SMO in crystallography experiments. In vertebrates, hedgehog signalling coordinates endochondral ossification and balances osteoblast and osteoclast activation to maintain homeostasis. A recently discovered mutation of SMO in humans (SMOR173C ) is predicted to alter cholesterol binding and is associated with a higher risk of hip OA. Functional studies in mice and human tissue analysis provide evidence that hedgehog signalling is pathologically activated in chondrocytes of osteoarthritic cartilage.

Prostate secretory protein 94 inhibits sterol binding and export by the mammalian CAP protein CRISP2 in a calcium-sensitive manner

Members of the CAP protein superfamily are present in all kingdoms of life and have been implicated in many different processes, including pathogen defense, immune evasion, sperm maturation, and cancer progression. Most CAP proteins are secreted glycoproteins and share a unique conserved αβα sandwich fold. The precise mode of action of this class of proteins, however, has remained elusive. Saccharomyces cerevisiae has three CAP family members, termed pathogen related in yeast (Pry). We have previously shown that Pry1 and Pry2 export sterols in vivo and that they bind sterols in vitro. This sterol binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with prostate secretory protein of 94 amino acids (PSP94), another major protein component in the seminal plasma. Here we examine whether the interaction between CRISP proteins and PSP94 affects the sterol binding function of CAP family members. We show that coexpression of PSP94 with CAP proteins in yeast abolished their sterol export function and the interaction between PSP94 and CAP proteins inhibits sterol binding in vitro. In addition, mutations that affect the formation of the PSP94–CRISP2 heteromeric complex restore sterol binding. Of interest, we found the interaction of PSP94 with CRISP2 is sensitive to high calcium concentrations. The observation that PSP94 modulates the sterol binding function of CRISP2 in a calcium-dependent manner has potential implications for the role of PSP94 and CRISP2 in prostate physiology and progression of prostate cancer.