Cryo-EM structure of human Wntless in complex with Wnt3a.

Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 2.2 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and a lateral opening. Wnt3a interacts with WLS at multiple interfaces, with the lipid moiety on Wnt3a traversing a hydrophobic tunnel of WLS transmembrane domain and inserting into membrane. A ß-hairpin of Wnt3a containing the conserved palmitoleoylation site interacts with WLS extensively, which is crucial for WLS-mediated Wnt secretion. The flexibility of the Wnt3a loop/hairpin regions involved in the multiple binding sites indicates induced fit might happen when Wnts are bound to different binding partners. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling.

Single-molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation.

Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of ß-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of dishevelled2 gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/µm2 to 0.54/µm2 Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.

Wnt3a Stimulation Promotes Primary Ciliogenesis through ß-Catenin Phosphorylation-Induced Reorganization of Centriolar Satellites.

Primary cilium is an antenna-like microtubule-based cellular sensing structure. Abnormal regulation of the dynamic assembly and disassembly cycle of primary cilia is closely related to ciliopathy and cancer. The Wnt signaling pathway plays a major role in embryonic development and tissue homeostasis, and defects in Wnt signaling are associated with a variety of human diseases, including cancer. In this study, we provide direct evidence of Wnt3a-induced primary ciliogenesis, which includes a continuous pathway showing that the stimulation of Wnt3a, a canonical Wnt ligand, promotes the generation of ß-catenin p-S47 epitope by CK1δ, and these events lead to the reorganization of centriolar satellites resulting in primary ciliogenesis. We have also confirmed the application of our findings in MCF-7/ADR cells, a multidrug-resistant tumor cell model. Thus, our data provide a Wnt3a-induced primary ciliogenesis pathway and may provide a clue on how to overcome multidrug resistance in cancer treatment.

Probing Interaction of Lipid-Modified Wnt Protein and Its Receptors by ELISA.

Wnts are lipid-modified proteins that regulate stem cell signaling via Frizzled receptors on the cell surface. Determination of binding interactions between lipid-modified Wnt proteins and their Frizzled receptors has been challenging due to the lack of availability of facile detection methods and technical hurdles associated with generating the relevant reagents. Here we report an enzyme-linked immunosorbent assay to measure the binding of a biotinylated form of lipid-modified Wnt3a to the extracellular cysteine-rich domain of Frizzled receptor. The method described herein is robust and rapid, uses minimum volumes of reagents, and can be conducted in a high-throughput format. Because of these attributes, the method could find utility in drug discovery applications such as characterizing the effect of pharmacological inhibitors on Wnt signaling without the need for sophisticated biophysical instrumentation.

Structural Evidence for a Role of the Multi-functional Human Glycoprotein Afamin in Wnt Transport.

Afamin, a human plasma glycoprotein and putative transporter of hydrophobic molecules, has been shown to act as extracellular chaperone for poorly soluble, acylated Wnt proteins, forming a stable, soluble complex with functioning Wnt proteins. The 2.1-Å crystal structure of glycosylated human afamin reveals an almost exclusively hydrophobic binding cleft capable of harboring large hydrophobic moieties. Lipid analysis confirms the presence of lipids, and density in the primary binding pocket of afamin was modeled as palmitoleic acid, presenting the native O-acylation on serine 209 in human Wnt3a. The modeled complex between the experimental afamin structure and a Wnt3a homology model based on the XWnt8-Fz8-CRD fragment complex crystal structure is compelling, with favorable interactions comparable with the crystal structure complex. Afamin readily accommodates the conserved palmitoylated serine 209 of Wnt3a, providing a structural basis how afamin solubilizes hydrophobic and poorly soluble Wnt proteins.

Characterization of secondary structure and lipid binding behavior of N-terminal saposin like subdomain of human Wnt3a.

Wnt signaling is essential for embryonic development and adult homeostasis in multicellular organisms. A conserved feature among Wnt family proteins is the presence of two structural domains. Within the N-terminal (NT) domain there exists a motif that is superimposable upon saposin-like protein (SAPLIP) family members. SAPLIPs are found in plants, microbes and animals and possess lipid surface seeking activity. To investigate the function of the Wnt3a saposin-like subdomain (SLD), recombinant SLD was studied in isolation. Bacterial expression of this Wnt fragment was achieved only when the core SLD included 82 NT residues of Wnt3a (NT-SLD). Unlike SAPLIPs, NT-SLD required the presence of detergent to achieve solubility at neutral pH. Deletion of two hairpin loop extensions present in NT-SLD, but not other SAPLIPs, had no effect on the solubility properties of NT-SLD. Far UV circular dichroism spectroscopy of NT-SLD yielded 50-60% α-helix secondary structure. Limited proteolysis of isolated NT-SLD in buffer and detergent micelles showed no differences in cleavage kinetics. Unlike prototypical saposins, NT-SLD exhibited weak membrane-binding affinity and lacked cell lytic activity. In cell-based canonical Wnt signaling assays, NT-SLD was unable to induce stabilization of ß-catenin or modulate the extent of ß-catenin stabilization induced by full-length Wnt3a. Taken together, the results indicate neighboring structural elements within full-length Wnt3a affect SLD conformational stability. Moreover, SLD function(s) in Wnt proteins appear to have evolved away from those commonly attributed to SAPLIP family members.

An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine.

Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O-acyl transferase family.

PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations.

AIM: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. METHODS: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. RESULTS: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. CONCLUSION: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.

Early Craniofacial Defects in Zebrafish That Have Reduced Function of a Wnt-Interacting Extracellular Matrix Protein, Tinagl1.

OBJECTIVE: Tinagl1 has a weak genetic association with craniosynostosis, but its functions in cartilage and bone development are unknown. Knockdown of Tinagl1 in zebrafish embryos allowed an initial characterization of its potential effects on craniofacial cartilage development and a test of whether these effects could involve Wnt signaling. RESULTS: Tinagl1 knockdown resulted in dose-dependent reductions and defects in ventral pharyngeal arch cartilages as well as the ethmoid plate, a zebrafish correlate to the palate. These defects could be correlated to reduced numbers of cranial neural crest cells in the pharyngeal arches and could be reproduced with comanipulation of Tinagl1 and Wnt3a by morpholino-based knockdown. CONCLUSIONS: These results suggest that Tinagl1 is required early in the proliferation or migration of cranial neural crest cells and that its effects are mediated via Wnt3a signaling. Because Wnt3a is among the Wnts that contribute to nonsyndromic cleft lip and cleft palate in mouse and man, further investigation of Tinagl1 may help to elucidate mechanisms underlying these disorders.

Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells.

BACKGROUND: Wnt proteins modulate development, stem cell fate and cancer through interactions with cell surface receptors. Wnts are cysteine-rich, glycosylated, lipid modified, two domain proteins that are prone to aggregation. The culprit responsible for this behavior is a covalently bound palmitoleoyl moiety in the N-terminal domain. RESULTS: By combining murine Wnt3a with phospholipid and apolipoprotein A-I, ternary complexes termed nanodisks (ND) were generated. ND-associated Wnt3a is soluble in the absence of detergent micelles and gel filtration chromatography revealed that Wnt3a co-elutes with ND. In signaling assays, Wnt3a ND induced ß-catenin stabilization in mouse fibroblasts as well as hematopoietic stem and progenitor cells (HSPC). Prolonged exposure of HSPC to Wnt3a ND stimulated proliferation and expansion of Lin(-) Sca-1(+) c-Kit(+) cells. Surprisingly, ND lacking Wnt3a contributed to Lin(-) Sca-1(+) c-Kit(+) cell expansion, an effect that was not mediated through ß-catenin. CONCLUSIONS: The data indicate Wnt3a ND constitute a water-soluble transport vehicle capable of promoting ex vivo expansion of HSPC.

Immobilized WNT Proteins Act as a Stem Cell Niche for Tissue Engineering.

The timing, location, and level of WNT signaling are highly regulated during embryonic development and for the maintenance of adult tissues. Consequently the ability to provide a defined and directed source of WNT proteins is crucial to fully understand its role in tissue development and to mimic its activity in vitro. Here we describe a one-step immobilization technique to covalently bind WNT3A proteins as a basal surface with easy storage and long-lasting activity. We show that this platform is able to maintain adult and embryonic stem cells while also being adaptable for 3D systems. Therefore, this platform could be used for recapitulating specific stem cell niches with the goal of improving tissue engineering.

Characterization of Tiki, a New Family of Wnt-specific Metalloproteases.

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn(2+)/Co(2+) but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core ß-sheet within an α+ß-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the ß-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.

Sequence and structural difference favors a distinct preference of Wnt3a binding with co-receptor LRP6.

Wnt signaling pathway plays a key role in a wide array of development and physiological processes. Wnt proteins interact with two different co-receptors LRP5/6 and ROR 2, leading to different signal transductions in the cell. Though the Wnt family of proteins has high sequence similarity the specificity for particular co-receptor is not well understood. The choice of pathway is attributed to the binding of Wnt complex to the co-receptor. Our current study is a novel approach using homology modeling, docking, and structural alignment to unravel the structural differences between Wnt3a and Wnt5b binding to LRP6. The conservation of a protruding loop has been identified in Wnt3a protein indicating an enhanced ability of Wnt3a to bind to LRP5/6 against its counter parts. The docking studies have further substantiated the findings. This could potentially help us design and develop novel inhibitors targeting Wnt3a-LRP6 complex in specific tissues or disease states.

Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity.

BACKGROUND: Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed. RESULTS: Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner. CONCLUSIONS: We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.

Identification of key residues and regions important for porcupine-mediated Wnt acylation.

Wnts comprise a family of lipid-modified, secreted signaling proteins that control embryogenesis, as well as tissue homeostasis in adults. Post-translational attachment of palmitoleate (C16:1) to a conserved Ser in Wnt proteins is catalyzed by Porcupine (Porcn), a member of the membrane bound O-acyltransferase (MBOAT) family, and is required for Wnt secretion and signaling. Moreover, genetic alterations in the PORCN gene lead to focal dermal hypoplasia, an X-linked developmental disorder. Despite its physiological importance, the biochemical mechanism governing Wnt acylation by Porcn is poorly understood. Here, we use a cell-based fatty acylation assay that is a direct readout of Porcn acyltransferase activity to perform structure-function analysis of highly conserved residues in Porcn and Wnt3a. In total, 16-point mutations in Porcn and 13 mutations in Wnt3a were generated and analyzed. We identified key residues within Porcn required for enzymatic activity, stability, and Wnt3a binding and mapped these active site residues to predicted transmembrane domain 9. Analysis of focal dermal hypoplasia-associated mutations in Porcn revealed that loss of enzymatic activity arises from altered stability. A consensus sequence within Wnt3a was identified (CXCHGXSXXCXXKXC) that contains residues that mediate Porcn binding, fatty acid transfer, and Wnt signaling. We also showed that Ser or Thr, but not Cys, can serve as a fatty acylation site in Wnt, establishing Porcn as an O-acyltransferase. This analysis sheds light into the mechanism by which Porcn transfers fatty acids to Wnt proteins and provides insight into the mechanisms of fatty acid transfer by MBOAT family members.

Disulfide bond requirements for active Wnt ligands.

Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired ß strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.

LRP6 dimerization through its LDLR domain is required for robust canonical Wnt pathway activation.

Canonical Wnt/ß-catenin signaling pathway plays important roles in multiple aspects of cellular responses in development and diseases. It is currently thought that Wnt receptor Frizzled (Frz) exists separately to Wnt coreceptors LRP5 and LRP6 (LRP5/6), and that Wnt-Frz-LRP5/6 triple complex formation bridged by Wnt ligand is needed for canonical pathway activation. We recently showed that Frz and LRP5/6 interact with each other in the absence of Wnt ligand binding and this interaction maintains the Frz-LRP5/6 complex in an inactive state. Here, we further show that Wnt ligand stimulation induces conformational change of the Frz-LRP6 complex and leads to hexamer formation containing the core LDLR domain-mediated LRP6 homodimer that is stabilized by two pairs of Wnt3a and Frz8, that is, Wnt3a-Frz8-LRP6-LRP6-Frz8-Wnt3a. This LDLR-mediated LRP6 dimerization is essential for robust canonical Wnt pathway activation. Our study thus suggests a previously unrecognized mode of receptor interaction in Wnt signal initiation.

Drugging a stem cell compartment using Wnt3a protein as a therapeutic.

The therapeutic potential of Wnt proteins has long been recognized but challenges associated with in vivo stability and delivery have hindered their development as drug candidates. By exploiting the hydrophobic nature of the protein we provide evidence that exogenous Wnt3a can be delivered in vivo if it is associated with a lipid vesicle. Recombinant Wnt3a associates with the external surface of the lipid membrane; this association stabilizes the protein and leads to prolonged activation of the Wnt pathway in primary cells. We demonstrate the consequences of Wnt pathway activation in vivo using a bone marrow engraftment assay. These data provide validation for the development of WNT3A as a therapeutic protein.

Anti-leprosy drug clofazimine inhibits growth of triple-negative breast cancer cells via inhibition of canonical Wnt signaling.

Research on existing drugs often discovers novel mechanisms of their action and leads to the expansion of their therapeutic scope and subsequent remarketing. The Wnt signaling pathway is of the immediate therapeutic relevance, as it plays critical roles in cancer development and progression. However, drugs which disrupt this pathway are unavailable despite the high demand. Here we report an attempt to identify antagonists of the Wnt-FZD interaction among the library of the FDA-approved drugs. We performed an in silico screening which brought up several potential antagonists of the ligand-receptor interaction. 14 of these substances were tested using the TopFlash luciferase reporter assay and four of them identified as active and specific inhibitors of the Wnt3a-induced signaling. However, further analysis through GTP-binding and ß-catenin stabilization assays showed that the compounds do not target the Wnt-FZD pair, but inhibit the signaling at downstream levels. We further describe the previously unknown inhibitory activity of an anti-leprosy drug clofazimine in the Wnt pathway and provide data demonstrating its efficiency in suppressing growth of Wnt-dependent triple-negative breast cancer cells. These data provide a basis for further investigations of the efficiency of clofazimine in treatment of Wnt-dependent cancers.

structural Studies of Wnts and identification of an LRP6 binding site.

Wnts are secreted growth factors that have critical roles in cell fate determination and stem cell renewal. The Wnt/ß-catenin pathway is initiated by binding of a Wnt protein to a Frizzled (Fzd) receptor and a coreceptor, LDL receptor-related protein 5 or 6 (LRP5/6). We report the 2.1 Å resolution crystal structure of a Drosophila WntD fragment encompassing the N-terminal domain and the linker that connects it to the C-terminal domain. Differences in the structures of WntD and Xenopus Wnt8, including the positions of a receptor-binding ß hairpin and a large solvent-filled cavity in the helical core, indicate conformational plasticity in the N-terminal domain that may be important for Wnt-Frizzled specificity. Structure-based mutational analysis of mouse Wnt3a shows that the linker between the N- and C-terminal domains is required for LRP6 binding. These findings provide important insights into Wnt function and evolution.