Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs

The seven-transmembrane-spanning receptors of the FZD_1–10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD₂, FZD₄, or FZD₅, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD_2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

Microsecond Time-Resolved Absorption Spectroscopy Used to Study CO Compounds of Cytochrome bd from Escherichia coli

Cytochrome bd is a tri-heme (b ₅₅₈, b ₅₉₅, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O₂ with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b ₅₉₅ in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b ₅₅₈ (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe²⁺-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b ₅₅₈ (τ∼0.2–3 ms), and finally migrates from heme d to heme b ₅₉₅ (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions.     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

Protein proximity networks and functional evaluation of the casein kinase 1 gamma family reveal unique roles for CK1γ3 in WNT signaling

Aberrant activation or suppression of WNT/β-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and “dark.” Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the β-catenin–dependent and β-catenin–independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated β-catenin–dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor–related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase–dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor–related protein 6 phosphorylation and β-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.     

Aberrant Activation of the WNT/β-Catenin Signaling Pathway in Lupus Nephritis

Objective

The canonical WNT pathway has been implicated as playing important roles in the pathogenesis of a variety of kidney diseases. Recently, WNT pathway activity was reported to be elevated in the renal tissue of a lupus mouse model. This study aimed to evaluate the potential role of the WNT pathway in the pathogenesis of human lupus nephritis.

Methods

The expression of β-catenin was evaluated in renal biopsy specimens from lupus nephritis patients and control kidney tissues by immunohistochemistry and western blotting. Real-time polymerase chain reaction (RT-PCR) was used to detect RNA expression of β-catenin, Dkk-1 and Axin2. Plasma concentrations of Dkk-1 were measured by ELISA.

Results

Immunohistochemistry and western blotting revealed increased expression of β-catenin in the kidneys of patients with lupus nephritis compared with control kidney tissues (p<0.05), accompanied by an increase in mRNA expression of β-catenin (p<0.01) and axin2 (p<0.05).β-catenin was significantly greater in LN patients without renal interstitial fibrosis compared with those with renal interstitial fibrosis (p<0.01) at the mRNA expression level; the increase in β-catenin mRNA positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury. Greater plasma Dkk-1 concentrations were found in LN patients compared with controls (p<0.05). Plasma Dkk-1 concentrations also correlated negatively with anti-dsDNA antibody levels and positively with serum C3 levels.

Conclusions

The canonical WNT/β-catenin signaling pathway was activated in lupus nephritis patients, accompanied by an increase in plasma levels of Dkk-1. Altered WNT/β-catenin signaling was related to the pathogenesis of lupus nephritis and might play a role in renal fibrosis.     

Dissociation of Increases in PGC-1α and Its Regulators from Exercise Intensity and Muscle Activation Following Acute Exercise

Muscle activation as well as changes in peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) following high-intensity interval exercise (HIIE) were examined in young healthy men (n  = 8; age, 21.9±2.2 yrs; VO₂peak, 53.1±6.4 ml/min/kg; peak work rate, 317±23.5 watts). On each of 3 visits HIIE was performed on a cycle ergometer at a target intensity of 73, 100, or 133% of peak work rate. Muscle biopsies were taken at rest and three hours after each exercise condition. Total work was not different between conditions (∼730 kJ) while average power output (73%, 237±21; 100%, 323±26; 133%, 384±35 watts) and EMG derived muscle activation (73%, 1262±605; 100%, 2089±737; 133%, 3029±1206 total integrated EMG per interval) increased in an intensity dependent fashion. PGC-1α mRNA was elevated after all three conditions (p<0.05), with a greater increase observed following the 100% condition (∼9 fold, p<0.05) compared to both the 73 and 133% conditions (∼4 fold). When expressed relative to muscle activation, the increase in PGC-1α mRNA for the 133% condition was less than that for the 73 and 100% conditions (p<0.05). SIRT1 mRNA was also elevated after all three conditions (∼1.4 fold, p<0.05), with no difference between conditions. These findings suggest that intensity-dependent increases in PGC-1α mRNA following submaximal exercise are largely due to increases in muscle recruitment. As well, the blunted response of PGC-1α mRNA expression following supramaximal exercise may indicate that signalling mediated activation of PGC-1α may also be blunted. We also indentify that increases in PDK4, SIRT1, and RIP140 mRNA following acute exercise are dissociated from exercise intensity and muscle activation, while increases in EGR1 are augmented with supramaximal HIIE (p<0.05).     

WNT/β-Catenin Signaling Regulates Multiple Steps of Myogenesis by Regulating Step-Specific Targets

Molecules involved in WNT/β-catenin signaling show specific spatiotemporal expression and play vital roles in myogenesis; however, it is still largely unknown how WNT/β-catenin signaling regulates each step of myogenesis. Here, we show that WNT/β-catenin signaling can control diverse biological processes of myogenesis by regulating step-specific molecules. In order to identify the temporally specific roles of WNT/β-catenin signaling molecules in muscle development and homeostasis, we used in vitro culture systems for both primary mouse myoblasts and C2C12 cells, which can differentiate into myofibers. We found that a blockade of WNT/β-catenin signaling in the proliferating cells decreases proliferation activity, but does not induce cell death, through the regulation of genes cyclin A2 (Ccna2) and cell division cycle 25C (Cdc25c). During muscle differentiation, the inhibition of WNT/β-catenin signaling blocks myoblast fusion through the inhibition of the Fermitin family homolog 2 (Fermt2) gene. Blocking WNT/β-catenin signaling in the well-differentiated myofibers results in the failure of maintenance of their structure by disruption of cadherin/β-catenin/actin complex formation, which plays a crucial role in connecting a myofiber''s cytoskeleton to the surrounding extracellular matrix. Thus, our results indicate that WNT/β-catenin signaling can regulate multiple steps of myogenesis, including cell proliferation, myoblast fusion, and homeostasis, by targeting step-specific molecules.     

Effects of Hydrophobic Macromolecular Crowders on Amyloid β (16–22) Aggregation

In Alzheimer’s disease (AD), the amyloid β (Aβ) peptide aggregates in the brain to form progressively larger oligomers, fibrils, and plaques. The aggregation process is strongly influenced by the presence of other macromolecular species, called crowders, that can exert forces on the proteins. One very common attribute of macromolecular crowders is their hydrophobicity. We examined the effect of hydrophobic crowders on protein aggregation by using discontinuous molecular dynamics (DMD) simulations in combination with an intermediate resolution protein model, PRIME20. The systems considered contained 48 Aβ (16–22) peptides and crowders with diameters of 5 Å, 20 Å, and 40 Å, represented by hard spheres or spheres with square-well/square-shoulder interactions, at a crowder volume fraction of ϕ = 0.10. Results show that low levels of crowder hydrophobicity are capable of increasing the fibrillation lag time and high levels of crowder hydrophobicity can fully prevent the formation of fibrils. The types of structures that remain during the final stages of the simulations are summarized in a global phase diagram that shows fibril, disordered oligomer, or β-sheet phases in the space spanned by crowder size and crowder hydrophobicity. In particular, at high levels of hydrophobicity, simulations with 5 Å crowders result in only disordered oligomers and simulations with 40 Å crowders result in only β-sheets. The presence of hydrophobic crowders reduces the antiparallel β-sheet content of fibrils, whereas hard sphere crowders increase it. Finally, strong hydrophobic crowders alter the secondary structure of the Aβ (16–22) monomers, bending them into a shape that is incapable of forming ordered β-sheets or fibrils. These results qualitatively agree with previous theoretical and experimental work.     

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of β-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1β, and tumor necrosis factor alpha (TNF-α). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.     

Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis

WNT signaling plays multiple roles in skeletal myogenesis during gestation and postnatal stages. The R-spondin (RSPO) family of secreted proteins and their cognate receptors, members of leucine-rich repeat-containing G protein-coupled receptor (LGR) family, have emerged as new regulatory components of the WNT signaling pathway. We previously showed that RSPO2 promoted myogenic differentiation via activation of WNT/β-catenin signaling in mouse myoblast C2C12 cells in vitro. However, the molecular mechanism by which RSPO2 regulates myogenic differentiation is unknown. Herein, we show that depletion of the LGR4 receptor severely disrupts myogenic differentiation and significantly diminishes the response to RSPO2 in C2C12 cells, showing a requirement of LGR4 in RSPO signaling during myogenic differentiation. We identify the transforming growth factor β (TGF-β) antagonist follistatin (Fst) as a key mediator of RSPO-LGR4 signaling in myogenic differentiation. We further demonstrate that Fst is a direct target of the WNT/β-catenin pathway. Activation and inactivation of β-catenin induced and inhibited Fst expression, respectively, in both C2C12 cells and mouse embryos. Specific TCF/LEF1 binding sites within the promoter and intron 1 region of the Fst gene were required for RSPO2 and WNT/β-catenin-induced Fst expression. This study uncovers a molecular cross talk between WNT/β-catenin and TGF-β signaling pivotal in myogenic differentiation.     

Hydration-State Change of Horse Heart Cytochrome c Corresponding to Trifluoroacetic-Acid-Induced Unfolding

We investigate the hydration state of horse-heart cytochrome c (hh cyt c) in the unfolding process induced by trifluoroacetic acid (TFA). The conformation of hh cyt c changes from the native (N) state (2.9 < pH < 6.0) to the acid-unfolded (U_A) state (1.7 < pH < 2.0) to the acid-induced molten globule (A) state (pH ∼1.2). Hydration properties of hh cyt c during this process are measured at 20°C by high-resolution dielectric relaxation (DR) spectroscopy, UV-vis absorbance, and circular dichroism spectroscopy. Constrained water of hh cyt c is observed at every pH as an ∼5-GHz Debye component (DC) (DR time, τ_D ∼30 ps) and its DR amplitude (DRA) is increased by 77% upon N-to-U_A transition, when pH changes from 6.0 to 2.0. Even in the N state, the DRA of the constrained-water component is found to be increased by 22% with decreasing pH from 6.0 to 2.9, suggesting an increase in the accessible surface area of native hh cyt c. Moreover, hypermobile water around native hh cyt c is detected at pH 6.0 as a 19-GHz DC (τ_D ∼ 8.4 ps < τ_DW = 9.4 ps), but is not found at other pH values. The DRA signal of constrained water is found to return to the pH 2.9 (N-state) level upon U_A-to-A transition. Fast-response water (slightly slower than bulk) around A-state hh cyt c is detected at pH 1.2, and this suggests some accumulation of TFA⁻ ions around the peptide chain. Thus, this high-resolution DR spectroscopy study reveals that hh cyt c exhibits significant hydration-state change in the TFA-unfolding process.     

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) Effect on Glucose Production,but Not Energy Metabolism,Is Independent of Hepatic AMPK in Vivo

Metabolic stress, as well as several antidiabetic agents, increases hepatic nucleotide monophosphate (NMP) levels, activates AMP-activated protein kinase (AMPK), and suppresses glucose production. We tested the necessity of hepatic AMPK for the in vivo effects of an acute elevation in NMP on metabolism. 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; 8 mg·kg⁻¹·min⁻¹)-euglycemic clamps were performed to elicit an increase in NMP in wild type (α1α2^lox/lox) and liver-specific AMPK knock-out mice (α1α2^lox/lox + Albcre) in the presence of fixed glucose. Glucose kinetics were equivalent in 5-h fasted α1α2^lox/lox and α1α2^lox/lox + Albcre mice. AMPK was not required for AICAR-mediated suppression of glucose production and increased glucose disappearance. These results demonstrate that AMPK is unnecessary for normal 5-h fasting glucose kinetics and AICAR-mediated inhibition of glucose production. Moreover, plasma fatty acids and triglycerides also decreased independently of hepatic AMPK during AICAR administration. Although the glucoregulatory effects of AICAR were shown to be independent of AMPK, these studies provide in vivo support for the AMPK energy sensor paradigm. AICAR reduced hepatic energy charge by ∼20% in α1α2^lox/lox, which was exacerbated by ∼2-fold in α1α2^lox/lox + Albcre. This corresponded to a ∼6-fold rise in AMP/ATP in α1α2^lox/lox + Albcre. Consistent with the effects on adenine nucleotides, maximal mitochondrial respiration was ∼30% lower in α1α2^lox/lox + Albcre than α1α2^lox/lox livers. Mitochondrial oxidative phosphorylation efficiency was reduced by 25%. In summary, these results demonstrate that the NMP capacity to inhibit glucose production in vivo is independent of liver AMPK. In contrast, AMPK promotes mitochondrial function and protects against a more precipitous fall in ATP during AICAR administration.     

The Structure of the Karrikin-Insensitive Protein (KAI2) in Arabidopsis thaliana

KARRIKIN INSENSITIVE 2 (KAI2) is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P2₁ 2₁ 2₁ (a  = 63.57 Å, b  = 66.26 Å, c  = 78.25 Å) and P2₁ (a  = 50.20 Å, b  = 56.04 Å, c  = 52.43 Å, β  = 116.12°) to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

Spatio-temporal Model of Endogenous ROS and Raft-Dependent WNT/Beta-Catenin Signaling Driving Cell Fate Commitment in Human Neural Progenitor Cells

Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the model. Thus, our results provide both new insights and means to further our understanding of canonical WNT/β-catenin signaling and the role of ROS as intracellular signaling mediator.