Inhibition of the Functional Interplay between Endoplasmic Reticulum (ER) Oxidoreduclin-1α (Ero1α) and Protein-disulfide Isomerase (PDI) by the Endocrine Disruptor Bisphenol A

Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b′ domain, preventing PDI from binding to unfolded proteins. The b′ domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b′ domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.     

Human OS-9, a Lectin Required for Glycoprotein Endoplasmic Reticulum-associated Degradation, Recognizes Mannose-trimmed N-Glycans

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of α1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER α1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.Recognition and sorting of improperly folded proteins is essential to cell survival, and hence, an elaborate quality control system is found in cells. ER⁴ quality control is well characterized with respect to the N-linked oligosaccharides regulating the folding and degradation of newly synthesized proteins in the ER (1). Immediately after polypeptides enter the ER, Glc₃Man₉GlcNAc₂ (G3M9) precursor oligosaccharides are covalently attached and subsequently processed. Terminally misfolded proteins are removed from the ER by the ERAD machinery (1–4). Aberrant conformers are recognized, retrotranslocated to the cytosol, and degraded by the ubiquitin-proteasome system (5, 6). Processing of mannose residues from the N-linked oligosaccharides acts as a timer for the recognition of misfolded glycoproteins in the ER lumen (1, 7). ER α1,2-mannosidase I (ER ManI) in mammals and ER α-mannosidase in yeast preferentially trim mannose residues from the middle branch of N-glycans, generating the Man₈GlcNAc₂ (M8) isomer B (M8B) (8). In mammals, further mannose processing is required as a signal for degradation (1, 9, 10), whereas the presence of M8B is sufficient to signal degradation in yeast (11). The postulated lectin EDEMs in mammals, their yeast homolog Htm1p/Mnl1p, and the yeast MRH domain-containing lectin Yos9p have all been proposed to recognize glycoproteins targeted for degradation (12).The role of Yos9p in glycoprotein ERAD was identified using a genetic screen in Saccharomyces cerevisiae (13). Yos9p, a homolog of hOS-9, contains an MRH domain (14) and functions as a lectin. Yos9p recognizes substrates of the ERAD-lumenal pathway (15–17), generating a large ER membrane complex containing the Hrd1p-Hrd3p ubiquitin ligase core complex (18–20). The M8B and Man₅GlcNAc₂ (M5) N-glycans are predicted to function as ligands for Yos9p (17). Bipartite recognition of both glycan and polypeptide by Yos9p has also been reported (15).Recent studies revealed that two mammalian MRH domain-containing lectins, OS-9 and XTP3-B, are ER luminal proteins involved in ER quality control and form a large complex containing the HRD1-SEL1L ubiquitin-ligase in the ER membrane (21–24). The components of the complex are similar to yeast, suggesting evolutionary conservation, although the molecular mechanisms underlying the role of OS-9 and XTP3-B remain elusive. Studies using lectin mutants have suggested that the MRH domains are required not for binding to ERAD substrates but for interactions with SEL1L (21), which has multiple N-glycans (25, 26). Additionally, lectin activity appears to be dispensable for hOS-9 binding to misfolded glycoproteins (21, 24). Thus, to understand the role of hOS-9 in the ER quality control pathway, the specific carbohydrate structures recognized by the hOS-9 MRH domain need to be identified, and the requirement of the lectin domain in substrate recognition needs to be determined.In the present study we demonstrate that the lectin activity of hOS-9 is required for enhancement of glycoprotein ERAD. We identified the N-glycan structures recognized by the recombinant hOS-9 MRH domain in vitro by frontal affinity chromatography (FAC). Using a model ERAD substrate, NHK (27), we show that the ability of hOS-9 to enhance ERAD in vivo depends on the oligosaccharides present on NHK, consistent with the FAC results.     

PKCδ and ε regulate the morphological integrity of the ER-Golgi intermediate compartment (ERGIC) but not the anterograde and retrograde transports via the Golgi apparatus

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.     

Activation of myeloid dendritic cells by deoxynucleic acids from Cordyceps sinensis via a Toll-like receptor 9-dependent pathway

The mechanism by which host cells recognize Cordyceps sinensis, a Chinese herbal medicine that is known to exhibit immunomodulating activity, remains poorly understood. In this study, we investigated whether the DNA of this fungus could activate mouse bone marrow-derived dendritic cells (BM-DCs). Upon stimulation with C. sinensis DNA, BM-DCs released IL-12p40 and TNF-α and expressed CD40. Cytokine production and CD40 expression were attenuated by chloroquin and bafilomycin A. Activation of BM-DCs by C. sinensis DNA was almost completely abrogated in TLR9KO mice. According to a luciferase reporter assay, C. sinensis DNA activated NF-κB in HEK293T cells transfected with the TLR9 gene. Finally, a confocal microscopic analysis showed that C. sinensis DNA was co-localized with CpG-ODN and partly with TLR9 and LAMP-1, a late endosomal marker, in BM-DCs. Our results demonstrated that C. sinensis DNA caused activation of BM-DCs in a TLR9-dependent manner.     

An RNA Interference Screen for Genes Required to Shape the Anteroposterior Compartment Boundary in Drosophila Identifies the Eph Receptor

The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. Here, we report on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.     

Role of disulfide bonds in goose-type lysozyme

The role of the two disulfide bonds (Cys4-Cys60 and Cys18-Cys29) in the activity and stability of goose-type (G-type) lysozyme was investigated using ostrich egg-white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild-type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild-type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G-type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys-free mutant was downshifted by about 20 degrees C as compared with that of the wild-type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G-type lysozyme.     

Appropriate preoperative membranous urethral length predicts recovery of urinary continence after robot-assisted laparoscopic prostatectomy

Purpose

We investigated that preoperative membranous urethral length (MUL) would be associated with the recovery of urinary continence after robot-assisted laparoscopic prostatectomy (RALP).

Patients and methods

We studied 204 patients who underwent RALP between May 2013 and March 2016. All patients underwent pelvic magnetic resonance imaging (MRI) preoperatively to measure MUL. Urinary continence was defined as the use of one pad or less (safety pad). The 204 patients were divided into two groups: continence group, those who achieved recovery of continence at 3, 6, and 12?months after RALP, and incontinence group, those who did not. We retrospectively analyzed the patients in terms of preoperative clinical factors including age, body mass index (BMI), estimated prostate volume, neurovascular bundle salvage, history of preoperative hormonal therapy, and MUL.

Results

The safety pad use rate was 69.6%, 86.9%, and 91.1% at 3, 6, and 12?months, respectively. On univariate and multivariate analyses, MUL were significant factors in every term of recovery of urinary continence in both groups. According to the receiver operating characteristic (ROC) curve analysis, the preoperative MUL that could best predict early recovery of urinary continence at 3 months after RALP was 12?mm.

Conclusions

We suggest that preoperative MUL >?12?mm would be a predictor of early recovery of urinary continence after RALP.

     

Haploid plants carrying a sodium azide-induced mutation (fdr1) produce fertile pollen grains due to first division restitution (FDR) in maize (Zea mays L.)

Key message

We induced a fdr1 mutation in maize which makes haploid plants male fertile due to first division restitution; the optimum sodium azide treatment on maize kernels has been identified.

Abstract

Sodium azide mutagenesis experiments were performed on haploid and diploid maize plants. Kernels with haploid embryos of maize inbred line B55 were induced by pollinating with RWS pollen. These kernels were treated with 0.2, 0.5, or 1.0 mM sodium azide solution for 2 h. The 0.5 mM solution was optimal for inducing numerous albino sectors on the treated plants without significant damage. Kernels of a maize hybrid, Oh43 × B55, were treated with sodium azide solutions at concentrations of 1.5, 2.0, 2.5, and 3.0 mM. Haploids were generated by pollinating RWS pollen. The highest rate of chlorophyll mutations in seedlings (15.3 % [13/85]) was recorded with the 2.5 mM concentration. A mutated haploid plant (PP1-50) with higher pollen fertility was isolated during the experiments. This haploid plant produced four kernels on the ear after selfing. These kernels were germinated and produced ears with full seed set after selfing. The haploid plants induced from PP1-50 diploids also exhibited high pollen fertility. In situ hybridization studies showed that meiocytes in PP1-50 haploid anthers underwent first division restitution at a rate of 48 % and produced equally divided dyads. We designated the genetic factor responsible for this high pollen fertility as fdr1. PP1-50 haploid ears exhibited high levels of sterility, as seen for regular haploids. Diploid PP1-50 meiocytes in the anther underwent normal meiosis, and all selfed progenies were normal diploids. We concluded that the fdr1 phenotype is only expressed in the anthers of haploid plants and not in the anthers of diploid plants.