The four R-spondin (Rspo) proteins are secreted agonists of Wnt signalling in vertebrates, functioning in embryogenesis and adult stem cell biology. Through ubiquitination and degradation of Wnt receptors, the transmembrane E3 ubiquitin ligase ZNRF3 and related RNF43 antagonize Wnt signalling. Rspo ligands have been reported to inhibit the ligase activity through direct interaction with ZNRF3 and RNF43. Here we report multiple crystal structures of the ZNRF3 ectodomain (ZNRF3 ecto ), a signalling-competent Furin1-Furin2 (Fu1-Fu2) fragment of Rspo2 (Rspo2 Fu1-Fu2 ), and Rspo2 Fu1-Fu2 in complex with ZNRF3 ecto , or RNF43 ecto . A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3 ecto and RNF43 ecto surface. Rspo binding enhances dimerization of ZNRF3 ecto but not of RNF43 ecto .Comparison of the four Rspo proteins, mutants and chimeras in biophysical and cellular assays shows that their signalling potency depends on their ability to recruit ZNRF3 or RNF43 via Fu1 into a complex with LGR receptors, which interact with Rspo via Fu2.

The functional diversity of large conductance Ca 2؉ -and voltage-dependent K ؉ (BK) channels arises mainly from co-assembly of the pore-forming mSlo ␣ subunits with four tissueenriched auxiliary ␤ subunits. The structural basis of the interaction between ␣ subunits with ␤ subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and h␤4 subunits together formed a "helmet" containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a K d ‫؍‬ 17 nM, suggesting that the h␤4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the ␣ subunits that could "permanently" block 15% of mSlo؉h␤4 currents in the presence of 100 nM ChTX. These findings provide a better understanding of the diverse interactions between ␣ and ␤ subunits and will improve the design of channel inhibitors.It is well known that functional diversity of large conductance Ca 2ϩ -and voltage-dependent potassium (BK) channels arises mainly from co-expression of the pore-forming ␣ subunits with the tissue-enriched auxiliary ␤ subunits (1-10). Four mammalian ␤ genes (␤1-␤4) are responsible for a variety of the kinetic and pharmacological characteristics of BK channels in native tissues, even though they share sequence homology. For instance, the brain-enriched h␤4 subunits not only alter the conductance-voltage curve of mSlo channels but also slow both the activation and deactivation time courses, suggesting that ␤4 plays a critical role in the regulation of neuronal excitability and neurotransmitter release (9). Furthermore, results from experiments with a ␤4 knock-out showed reduced dentate gyrus excitability and protection against temporal lobe seizures (11).Natural venomous peptides have been used widely to probe the pore conformation of potassium channels. One particular scorpion toxin, charybdotoxin (ChTX), 4 was used to probe the pore structure of BK channels. It is known that the ␣ϩh␤1 has a K d for ChTX similar to that of mSlo channels (4). However, BK channels associating with the ␤2 or ␤3 subunits usually have about 30-fold lower sensitivity to ChTX in equilibrium (3, 4), whereas the ␣ϩh␤4 channel has about 1000-fold slower association with the toxin (7). Is there a common mechanism working for all of four ␤ genes?To explore the toxin resistance mechanism of h␤4 subunits, Jin et al. (12) investigated how the N-linked glycosylation of the h␤4 subunit modulated the toxin sensitivity of hSloϩh␤4 channels. They confirmed that the double glycosylation site mutated in h␤4 showed reduced protection of the channel against toxin blockade as compared with the hSlo channel co-expressed with the wild type h␤4 subunits. Another study is in regard to the remo...

Dysregulated physical stresses are generated during tumorigenesis that affect the surrounding compliant tissues including adipocytes. However, the effect of physical stressors on the behavior of adipocytes and their cross-talk with tumor cells remain elusive. Here, we demonstrate that compression of cells, resulting from various types of physical stresses, can induce dedifferentiation of adipocytes via mechanically activating Wnt/β-catenin signaling. The compression-induced dedifferentiated adipocytes (CiDAs) have a distinct transcriptome profile, long-term self-renewal, and serial clonogenicity, but do not form teratomas. We then show that CiDAs notably enhance human mammary adenocarcinoma proliferation both in vitro and in a xenograft model, owing to myofibrogenesis of CiDAs in the tumor-conditioned environment. Collectively, our results highlight unique physical interplay in the tumor ecosystem; tumor-induced physical stresses stimulate de novo generation of CiDAs, which feedback to tumor growth.

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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 β-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.

UBIAD1 plays critical roles in physiology including vitamin K and CoQ10 biosynthesis as well as pathophysiology including dyslipimedia-induced SCD (Schnyder’s corneal dystrophy), Parkinson’s disease, cardiovascular disease and bladder carcinoma. Since the subcellular localization of UBIAD1 varies in different cell types, characterization of the exact subcellular localization of UBIAD1 in specific human disease is vital for understanding its molecular mechanism. As UBIAD1 suppresses bladder carcinoma, we studied its subcellular localization in human bladder carcinoma cell line T24. Since fluorescent images of UBIAD1-EGFP in T24, human prostate cancer cell line PC-3, human embryonic kidney cell line HEK293 and human hepatocyte cell line L02 are similar, these four cell lines were used for present study. Using a combination of fluorescent microscopy and immunohistochemistry, it was found that UBIAD1 localized on the Golgi and endoplasmic reticulum (ER), but not on the plasma membrane, of T24 and HEK293 cells. Using scanning electron microscopy and western blot analysis, we found that UBIAD1 is enriched in the Golgi fraction extracted from the L02 cells, verifying the Golgi localization of UBAID1. Site-directed mutagenesis showed that the RPWS motif, which forms an Arginine finger on the UBIAD1 N terminus, serves as the Golgi retention signal. With both cycloheximide and brefeldin A inhibition assays, it was shown that UBIAD1 may be transported from the endoplasmic reticulum (ER) to the Golgi by a COPII-mediated mechanism. Based upon flow cytometry analysis, it is shown that mutation of the RPWS motif reduced the UBIAD1-induced apoptosis of T24 cells, indicating that the proper Golgi localization of UBIAD1 influences its tumor suppressant activity. This study paves the way for further understanding the molecular mechanism of UBIAD1 in human diseases.

The auxiliary β subunits of large-conductance Ca2+-activated K+ (BK) channels greatly contribute to the diversity of BK (mSlo1 α) channels, which is fundamental to the adequate function in many tissues. Here we describe a functional element of the extracellular segment of hβ2 auxiliary subunits that acts as the positively charged rings to modify the BK channel conductance. Four consecutive lysines of the hβ2 extracellular loop, which reside sufficiently close to the extracellular entryway of the pore, constitute three positively charged rings. These rings can decrease the extracellular K+ concentration and prevent the Charybdotoxin (ChTX) from approaching the extracellular entrance of channels through electrostatic mechanism, leading to the reduction of K+ inflow or the outward rectification of BK channels. Our results demonstrate that the lysine rings formed by the hβ2 auxiliary subunits influences the inward current of BK channels, providing a mechanism by which current can be rapidly diminished during cellular repolarization. Furthermore, this study will be helpful to understand the functional diversity of BK channels contributed by different auxiliary β subunits.

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