Tributyltin-binding protein type 1 (fish acid glycoprotein) is a potential gatekeeper of ethinylestradiol action in fish.

Tributyltin (TBT)-binding protein type 1 in Japanese medaka (Oryzias latipes) (O.latTBT-bp1) is a fish lipocalin implicated in TBT binding and detoxification. We purified recombinant O.latTBT-bp1 (rO.latTBT-bp1; ca. 30 kDa) by using a baculovirus expression system and His- and Strep-tag chromatography process. Then, we examined O.latTBT-bp1 binding to several endo/exogenous steroid hormones by means of competitive binding assay. The dissociation constants for the binding of rO.latTBT-bp1 to DAUDA and ANS, two fluorescent ligands of lipocalin, were 7.06 and 13.6 µM, respectively. Multiple model validations indicated that a single-binding-site model was the most appropriate for evaluating rO.latTBT-bp1 binding. In the competitive binding assay, testosterone, 11-ketotestosterone, and 17ß-estradiol were each bound by rO.latTBT-bp1; rO.latTBT-bp1 showed the strongest affinity for testosterone (inhibition constant, Ki = 3.47 µM). Endocrine-disrupting chemical (synthetic steroid) also bound to rO.latTBT-bp1; the affinity for ethinylestradiol (Ki = 9.29 µM) was stronger than that for 17ß-estradiol (Ki = 30.0 µM). To determine the function of O.latTBT-bp1, we produced TBT-bp1 knockout medaka (TBT-bp1 KO), which we exposed to ethinylestradiol for 28 days. After exposure, the number of papillary processes in TBT-bp1 KO genotypic male medaka was significantly fewer (3.5), compared to that in wild-type male medaka (22). Thus, TBT-bp1 KO medaka were more sensitive to the anti-androgenic effects of ethinylestradiol than wild-type medaka. These results indicate that O.latTBT-bp1 may bind to steroids and act as a gatekeeper of ethinylestradiol action by regulating the androgen-estrogen balance.

Transdermal Transmission Blocking Vaccine for Malaria using a Solid-in-Oil Dispersion.

Malaria is a mosquito-borne infectious disease that is widespread in developing countries. Malaria vaccines are important in efforts to eradicate malaria; however, vaccines are usually administered by injection, which requires medical personnel and has a risk of causing infection. Transdermal vaccines can be administered without damaging the skin and thus are ideal for the prevention of malaria. However, the stratum corneum forms a "brick and mortar" like structure in which stratum corneum cells are embedded in a hydrophobic matrix composed of lipids, which strongly inhibits the permeation of hydrophilic substances. In the present study, we designed a transdermal vaccine against vivax malaria using a solid-in-oil (S/O) dispersion. The S/O dispersion of a transmission blocking vaccine candidate, Pvs25 from Plasmodium vivax, showed higher skin penetration than that of the aqueous solution. Mice immunized with the S/O dispersion generated antibodies at similar titers as the mice immunized by injection, over the mid- to long-term. These results provide information for the development of transdermally administered malaria vaccines toward the eradication of malaria.

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis.

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

The benefits of the earlier use of sacubitril/valsartan in de novo heart failure with reduced ejection fraction patients.

AIMS: We evaluated the clinical outcomes and trajectory of cardiac reverse remodelling according to the timing of sacubitril/valsartan (Sac/Val) use in patients with heart failure (HF) with reduced ejection fraction (HFrEF). METHODS AND RESULTS: Patients with de novo HFrEF who used Sac/Val between June 2017 and October 2019 were retrospectively enrolled. Patients were grouped into the earlier use group (initiation of Sac/Val < 3 months after the first HFrEF diagnosis) and the later use group (initiation of Sac/Val ≥ 3 months after the first HFrEF diagnosis). Primary outcome was a composite of HF hospitalization and cardiac death. Secondary outcomes were HF hospitalization, cardiac death, all-cause death, significant ventricular arrhythmia (ventricular tachycardia or ventricular fibrillation), and echocardiographic evidence of cardiac reverse remodelling including left ventricular ejection fraction (LVEF) change during follow-up. Among 115 enrolled patients, 67 were classified in the earlier use group, and 48 were classified in the later use group. Mean period of HFrEF diagnosis to Sac/Val use was 52.1 ± 14.3 days in the earlier use group, and 201.8 ± 127.3 days in the later use group. During the median follow-up of 721 days, primary outcome occurred in 21 patients (18.3%). The earlier use group experienced significantly fewer primary outcome than the later use group (10.4% vs. 29.2%, P = 0.010). The Kaplan-Meier survival curve showed better event-free survival in the earlier use group than in the later use group (log rank = 0.017). There were no significant differences in cardiac death, all-cause death, and ventricular arrhythmia between two groups (1.5% vs. 2.1%, P = 0.811; 1.5% vs. 4.2%, P = 0.375; 3.0% vs. 0%, P = 0.227, respectively). Despite a significantly lower baseline LVEF in the earlier use group (21.3 ± 6.4% vs. 24.8 ± 7.9%, P = 0.012), an early prominent increase of LVEF was noted before 6 months (35.2 ± 11.9% vs. 27.8 ± 8.8%, P = 0.007). A delayed improvement of LVEF in the later use group resulted in similar LVEF at last follow-up in both groups (40.7 ± 13.4% vs. 39.4 ± 10.9%, P = 0.686). Although the trajectory of left ventricular remodelling showed similar pattern in two groups, left atrial (LA) reverse remodelling was less prominent in the later use group during the follow-up period (final LA volume index: 43.6 ± 14.3 mL/m2 vs. 55.2 ± 17.1 mL/m2 , P = 0.011). CONCLUSIONS: Earlier use of Sac/Val was related with better clinical outcome and earlier left ventricular reverse remodelling. Remodelling of LA was less prominent in the later use group implying delayed response in diastolic function.

Transcriptional Regulation of Hepatic Autophagy by Nuclear Receptors.

Autophagy is an adaptive self-eating process involved in degradation of various cellular components such as carbohydrates, lipids, proteins, and organelles. Its activity plays an essential role in tissue homeostasis and systemic metabolism in response to diverse challenges, including nutrient depletion, pathogen invasion, and accumulations of toxic materials. Therefore, autophagy dysfunctions are intimately associated with many human diseases such as cancer, neurodegeneration, obesity, diabetes, infection, and aging. Although its acute post-translational regulation is well described, recent studies have also shown that autophagy can be controlled at the transcriptional and post-transcriptional levels. Nuclear receptors (NRs) are in general ligand-dependent transcription factors consisting of 48 members in humans. These receptors extensively control transcription of a variety of genes involved in development, metabolism, and inflammation. In this review, we discuss the roles and mechanisms of NRs in an aspect of transcriptional regulation of hepatic autophagy, and how the NR-driven autophagy pathway can be harnessed to treat various liver diseases.

Basal Autophagy Is Necessary for A Pharmacologic PPARα Transactivation.

Autophagy is a conserved cellular process of catabolism leading to nutrient recycling upon starvation and maintaining tissue and energy homeostasis. Tissue-specific loss of core-autophagy-related genes often triggers diverse diseases, including cancer, neurodegeneration, inflammatory disease, metabolic disorder, and muscle disease. The nutrient-sensing nuclear receptors peroxisome proliferator-activated receptor α (PPARα) plays a key role in fasting-associated metabolisms such as autophagy, fatty acid oxidation, and ketogenesis. Here we show that autophagy defects impede the transactivation of PPARα. Liver-specific ablation of the Atg7 gene in mice showed reduced expression levels of PPARα target genes in response to its synthetic agonist ligands. Since NRF2, an antioxidant transcription factor, is activated in autophagy-deficient mice due to p62/SQSTM1 accumulation and its subsequent interaction with KEAP1, an E3 ubiquitin ligase. We hypothesize that the nuclear accumulation of NRF2 by autophagy defects blunts the transactivation of PPARα. Consistent with this idea, we find that NRF2 activation is sufficient to inhibit the pharmacologic transactivation of PPARα, which is dependent on the Nrf2 gene. These results reveal an unrecognized requirement of basal autophagy for the transactivation of PPARα by preventing NRF2 from a nuclear translocation and suggest a clinical significance of basal autophagy to expect a pharmacologic efficacy of synthetic PPARα ligands.

Transcriptional Control of Trpm6 by the Nuclear Receptor FXR.

Farnesoid x receptor (FXR) is a nuclear bile acid receptor that belongs to the nuclear receptor superfamily. It plays an essential role in bile acid biosynthesis, lipid and glucose metabolism, liver regeneration, and vertical sleeve gastrectomy. A loss of the FXR gene or dysregulations of FXR-mediated gene expression are associated with the development of progressive familial intrahepatic cholestasis, tumorigenesis, inflammation, and diabetes mellitus. Magnesium ion (Mg2+) is essential for mammalian physiology. Over 600 enzymes are dependent on Mg2+ for their activity. Here, we show that the Trpm6 gene encoding a Mg2+ channel is a direct FXR target gene in the intestinal epithelial cells of mice. FXR expressed in the intestinal epithelial cells is absolutely required for sustaining a basal expression of intestinal Trpm6 that can be robustly induced by the treatment of GW4064, a synthetic FXR agonist. Analysis of FXR ChIP-seq data revealed that intron regions of Trpm6 contain two prominent FXR binding peaks. Among them, the proximal peak from the transcription start site contains a functional inverted repeat 1 (IR1) response element that directly binds to the FXR-RXRα heterodimer. Based on these results, we proposed that an intestinal FXR-TRPM6 axis may link a bile acid signaling to Mg2+ homeostasis.

Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.

Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System.

The tumor necrosis factor α (TNFα) has been employed as a promising reagent in treating autoimmunity and cancer diseases. To meet the substantial requirement of TNFα proteins, we report in this study that mature types of recombinant human and murine TNFα proteins are successfully expressed in the baculovirus expression system using silkworm larvae as hosts. The biological activities of purified products were verified in culture murine L929 cells, showing better performance over a commercial Escherichia coli-derived murine TNFα. By comparing the activity of purified TNFα with or without the tag removal, it is also concluded that the overall activity of purified TNFα cytokines could be further improved by the complete removal of C-terminal fusion tags. Collectively, our current attempt demonstrates an alternative platform for supplying high-quality TNFα products with excellent activities for further pharmaceutical and clinical trials.

Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration.

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca2+-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.

Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.

Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells.

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.

FXR-dependent Rubicon induction impairs autophagy in models of human cholestasis.

BACKGROUND & AIMS: Cholestasis comprises a spectrum of liver diseases characterized by the accumulation of bile acids. Bile acids and activation of the farnesoid X receptor (FXR) can inhibit autophagy, a cellular self-digestion process necessary for cellular homeostasis and regeneration. In mice, autophagy appears to be impaired in cholestasis and induction of autophagy may reduce liver injury. METHODS: Herein, we explored autophagy in human cholestasis in vivo and investigated the underlying molecular mechanisms in vitro. FXR chromatin immunoprecipitation-sequencing and qPCR were performed in combination with luciferase promoter studies to identify functional FXR binding targets in a human cholestatic liver sample. RESULTS: Autophagic processing appeared to be impaired in patients with cholestasis and in individuals treated with the FXR ligand obeticholic acid (OCA). In vitro, chenodeoxycholic acid and OCA inhibited autophagy at the level of autophagosome to lysosome fusion in an FXR-dependent manner. Rubicon, which inhibits autophago-lysosomal maturation, was identified as a direct FXR target that is induced in cholestasis and by FXR-agonistic bile acids. Genetic inhibition of Rubicon reversed the bile acid-induced impairment of autophagic flux. In contrast to OCA, ursodeoxycholic acid (UDCA), which is a non-FXR-agonistic bile acid, induced autophagolysosome formation independently of FXR, enhanced autophagic flux and was associated with reduced Rubicon levels. CONCLUSION: In models of human cholestasis, autophagic processing is impaired in an FXR-dependent manner, partly resulting from the induction of Rubicon. UDCA is a potent inducer of hepatic autophagy. Manipulating autophagy and Rubicon may represent a novel treatment concept for cholestatic liver diseases. LAY SUMMARY: Autophagy, a cellular self-cleansing process, is impaired in various forms of human cholestasis. Bile acids, which accumulate in cholestatic liver disease, induce Rubicon, a protein that inhibits proper execution of autophagy. Ursodeoxycholic acid, which is the first-line treatment option for many cholestatic liver diseases, induces hepatic autophagy along with reducing Rubicon.

Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain.

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system.

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.

Sulfisoxazole inhibits the secretion of small extracellular vesicles by targeting the endothelin receptor A.

Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology.

Expression, Purification, and Characterization of Recombinant Human α1-Antitrypsin Produced Using Silkworm-Baculovirus Expression System.

Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.

A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells.

Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (ß1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans.

Upregulation of neuronal astrocyte elevated gene-1 protects nigral dopaminergic neurons in vivo.

The role of astrocyte elevated gene-1 (AEG-1) in nigral dopaminergic (DA) neurons has not been studied. Here we report that the expression of AEG-1 was significantly lower in DA neurons in the postmortem substantia nigra of patients with Parkinson's disease (PD) compared to age-matched controls. Similarly, decreased AEG-1 levels were found in the 6-hydroxydopamine (6-OHDA) mouse model of PD. An adeno-associated virus-induced increase in the expression of AEG-1 attenuated the 6-OHDA-triggered apoptotic death of nigral DA neurons. Moreover, the neuroprotection conferred by the AEG-1 upregulation significantly intensified the neurorestorative effects of the constitutively active ras homolog enriched in the brain [Rheb(S16H)]. Collectively, these results demonstrated that the sustained level of AEG-1 as an important anti-apoptotic factor in nigral DA neurons might potentiate the therapeutic effects of treatments, such as Rheb(S16H) administration, on the degeneration of the DA pathway that characterizes PD.

Structure-function analyses of a stereotypic rheumatoid factor unravel the structural basis for germline-encoded antibody autoreactivity.

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called "stereotypic RFs," are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2-CH3 elbow in the canonical antigen-binding manner involving a large antigen-antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu432-His435 region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.