The HSP70-fused foot-and-mouth disease epitope elicits cellular and humoral immunity and drives broad-spectrum protective efficacy.

Current foot-and-mouth disease (FMD) vaccines have significant limitations, including side effects due to oil emulsions at the vaccination site, a narrow spectrum of protective efficacy, and incomplete host defenses mediated by humoral immunity alone. To overcome these limitations, new FMD vaccines must ensure improved safety with non-oil-based adjuvants, a broad spectrum of host defenses within/between serotypes, and the simultaneous induction of cellular and humoral immunity. We designed a novel, immune-potent, recombinant protein rpHSP70-AD that induces robust cellular immunity and elicits a broad spectrum of host defenses against FMD virus (FMDV) infections. We demonstrated that an oil emulsion-free vaccine containing rpHSP70-AD mediates early, mid-term, and long-term immunity and drives potent host protection against FMDV type O and A, suggesting its potential as an FMD vaccine adjuvant in mice and pigs. These results suggest a key strategy for establishing next-generation FMD vaccines, including novel adjuvants.

Expression system for production of bioactive compounds, recombinant human adiponectin, in the silk glands of transgenic silkworms.

Adiponectin is an adipocyte hormone involved in glucose and lipid metabolism. The aim of this study was to develop a human adiponectin expression system in transgenic silkworm using a human adiponectin expression vector. The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human adiponectin in the silk gland of transgenic silkworm, targeting vectors pB-A3-adiponectin-IRES-RFP and pB-Ser1-adiponectin-IRES-RFP were constructed and then introduced into the silkworm pupa. The transgenic silkworms were verified by PCR and then generated. The level of adiponectin in the transgenic silkworm was 6-10 ng/50 mg of freeze-dried powder, and western blotting using an antibody against human adiponectin demonstrated a specific band with a molecular weight of 30 kDa in the silkworm. These results showed that human adiponectin introduced into the silkworm genome was expressed successfully on a large-scale.

Impaired autophagy and delayed autophagic clearance of transforming growth factor ß-induced protein (TGFBI) in granular corneal dystrophy type 2.

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease characterized by a progressive age-dependent extracellular accumulation of transforming growth factor ß-induced protein (TGFBI). Corneal fibroblasts from GCD2 patients also have progressive degenerative features, but the mechanism underlying this degeneration remains unknown. Here we observed that TGFBI was degraded by autophagy, but not by the ubiquitin/proteasome-dependent pathway. We also found that GCD2 homozygous corneal fibroblasts displayed a greater number of fragmented mitochondria. Most notably, mutant TGFBI (mut-TGFBI) extensively colocalized with microtubule-associated protein 1 light chain 3ß (MAP1LC3B, hereafter referred to as LC3)-enriched cytosolic vesicles and CTSD in primary cultured GCD2 corneal fibroblasts. Levels of LC3-II, a marker of autophagy activation, were significantly increased in GCD2 corneal fibroblasts. Nevertheless, levels of SQSTM1/p62 and of polyubiquitinated protein were also significantly increased in GCD2 corneal fibroblasts compared with wild-type (WT) cells. However, LC3-II levels did not differ significantly between WT and GCD2 cells, as assessed by the presence of bafilomycin A 1, the fusion blocker of autophagosomes and lysosomes. Likewise, bafilomycin A 1 caused a similar change in levels of SQSTM1. Thus, the increase in autophagosomes containing mut-TGFBI may be due to inefficient fusion between autophagosomes and lysosomes. Rapamycin, an autophagy activator, decreased mut-TGFBI, whereas inhibition of autophagy increased active caspase-3, poly (ADP-ribose) polymerase 1 (PARP1) and reduced the viability of GCD2 corneal fibroblasts compared with WT controls. These data suggest that defective autophagy may play a critical role in the pathogenesis of GCD2.

Inhibition of TGFBIp expression by lithium: implications for TGFBI-linked corneal dystrophy therapy.

PURPOSE. The purpose of this study was to investigate the effects and molecular mechanisms of lithium on inhibition of TGFBIp expression as a potential therapy for TGFBI-linked corneal dystrophy. METHODS. Primary culture corneal fibroblasts were isolated from the corneas of healthy subjects and patients with granular corneal dystrophy type 2 (GCD2) with a homozygous mutation in TGFBI R124H. Levels of TGFBIp and its mRNA in corneal fibroblasts treated with various lithium (LiCl) concentrations were analyzed by Western blot, RT-PCR, and quantitative real-time PCR. RESULTS. LiCl treatment reduced the expression levels of normal and mutant TGFBIp in a dose- and a time-dependent manner. Furthermore, TGF-ß1-induced TGFBIp expression decreased by 35% and 67% after treatment with 5 mM and 10 mM LiCl, respectively. LiCl decreased the level of pSmad3 (S423/425) in a dose-dependent manner. Furthermore, LiCl increased the level of pGSK-3α/ß (S21/9) in a dose-dependent manner. Also observed was the interaction between GSK-3ß and Smad3, which was enhanced by lithium. In addition, Western blot analysis showed that the ratio of LC3-II/LC3-I in corneal fibroblasts increased after LiCl treatment. Cell viability at different doses was greater than 98%, indicating that LiCl did not induce significant corneal fibroblast death. Finally, the observed attenuating effects of LiCl on TGFBIp expression were not the results of cell death. CONCLUSIONS. The accumulation of mutant TGFBIp ultimately leads to the histopathologic and clinical manifestations associated with TGFBI-linked corneal dystrophy. These data strongly suggest that lithium may be used for the prevention or treatment of this disease.

Clinical findings and treatments of granular corneal dystrophy type 2 (avellino corneal dystrophy): a review of the literature.

OBJECTIVES: To review the literature about clinical findings and treatments of granular corneal dystrophy type 2 (GCD2). METHODS: Various literatures on clinical findings, exacerbations after refractive corneal surgery, and treatment modalities of GCD2 were reviewed. RESULTS: GCD2 is an autosomal dominant disease. Mutation of transforming growth factor beta-induced gene, TGFBI, or keratoepithelin gene in human chromosome 5 (5q31) is the key pathogenic process in patient with GCD2. Corneal trauma activates TGFBI and then it overproduces transforming growth factor beta-induced gene protein (TGFBIp), which is main component of the corneal opacity. Refractive corneal surgery is a popular procedure to correct refractive error worldwide. However, several cases about exacerbation of GCD2 after corneal refractive surgery such as photorefractive keratectomy, laser in situ keratomileusis, and laser epithelial keratomileusis have been reported. The opacities deteriorate patient's best-corrected visual acuity. Recurrence-free interval varies many factors such as the type of procedure the patient had received and the genotype of the patient. To treat the opacities in GCD2, phototherapeutic keratectomy, lamellar keratoplasty, deep lamellar keratoplasty, and penetrating keratoplasty (PKP) were used. However, the recurrence is still an unsolved problem. CONCLUSIONS: Perfect treatment of exacerbation after corneal surface ablation does not exist until now. To prevent exacerbation, refractive surgeons must do a careful preoperative examination of candidates in refractive surgeries.

Involvement of TGF-{beta} receptor- and integrin-mediated signaling pathways in the pathogenesis of granular corneal dystrophy II.

Purpose. The purpose of this study was to elucidate the pathophysiological process in primary cultured corneal fibroblasts (PCFs) from normal subjects and granular corneal dystrophy (GCD) II patients, by using cDNA microarrays. Methods. PCFs were isolated from the corneas of normal subjects and GCD II patients who were heterozygous and homozygous for the TGFBI R124H mutation. RNA was isolated from each sample, and gene expression profiles were analyzed with a cDNA microarray consisting of approximately 29,000 genes. Cell adhesion assays were performed to confirm the functionality of the detected gene expression profiles. Results. Twofold differences were detected in the expression of 555 genes between wild-type and homozygous GCD II PCFs. Of these, 319 genes were upregulated, and 236 genes were downregulated in the homozygous GCD II PCFs. The most abundant and consistent changes were observed in gene families encoding signal transduction pathways involving the TGF-beta receptor- and integrin-mediated signaling, cell differentiation and proliferation, immune responses, cell adhesion, extracellular matrix (ECM) proteolytic enzymes, cell cycle, cytoskeletal organization, mitochondrial energy metabolism, collagen catabolism, response to wounding, response to oxidative stress, and the ubiquitin-mediated proteasomal degradation pathway. Cell adhesion assays demonstrated that heterozygous and homozygous GCD II PCFs strongly attached to collagen-I, collagen-IV, fibronectin, and laminin, compared with wild-type cells. Conclusions. Alterations in the TGF-beta receptor- and integrin-mediated signaling pathway may play a key role in GCD II pathophysiology. If the novel factors identified in this study are involved in GCD II pathogenesis, they could assist in designing further studies to elucidate specific mechanisms of this disease.

Decreased catalase expression and increased susceptibility to oxidative stress in primary cultured corneal fibroblasts from patients with granular corneal dystrophy type II.

Granular corneal dystrophy type II (GCD II) is an autosomal dominant disorder characterized by age-dependent progressive accumulation of transforming growth factor-beta-induced protein (TGFBIp) deposits in the corneal stroma. Several studies have suggested that corneal fibroblasts may decline with age in response to oxidative stress. To investigate whether oxidative stress is involved in the pathogenesis of GCD II, we assayed antioxidant enzymes, oxidative damage, and susceptibility to reactive oxygen species-induced cell death in primary cultured corneal fibroblasts (PCFs) from GCD II patients and healthy subjects. We found elevated protein levels of Mn-superoxide dismutase, Cu/Zn-superoxide dismutase, glutathione peroxidase, and glutathione reductase, as well as increased CAT mRNA and decreased catalase protein in GCD II PCFs. Furthermore, catalase is down-regulated in normal PCFs transfected with transforming growth factor-beta-induced gene-h3. We also observed an increase in not only intracellular reactive oxygen species and H(2)O(2) levels, but also malondialdehyde, 4-hydroxynonenal, and protein carbonyls levels in GCD II PCFs. Greater immunoreactivity for malondialdehyde was observed in the corneal tissue of GCD II patients. In addition, we observed a decrease in Bcl-2 and Bcl-xL levels and an increase in Bax and Bok levels in GCD II PCFs. Finally, GCD II PCFs are more susceptible to H(2)O(2)-induced cell death. Together, these results suggest that oxidative damage induced by decreased catalase is involved in GCD II pathogenesis, and antioxidant agents represent a possible treatment strategy.

Corneal dystrophy-associated R124H mutation disrupts TGFBI interaction with Periostin and causes mislocalization to the lysosome.

The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-beta-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies.

Nitric oxide-dependent proteasomal degradation of cytochrome P450 2B proteins.

Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is down-regulated in primary cultures of rat hepatocytes after exposure to bacterial endotoxin (lipopolysaccharide) in a nitric oxide (NO) -dependent manner. In this study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed >60% after 6 h of treatment with interleukin-1beta (IL-1). This effect was NO-dependent, and treatment of cells with the NO donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione, and S-nitroso-N-acetylpenicillamine also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. The down-regulation by IL-1 or NO donors was abolished by treatments with the proteasome inhibitors MG132 and lactacystin that did not affect NO production. The calpain inhibitor E64-d or the lysosomal protease inhibitors NH(4)Cl and chloroquine did not attenuate the down-regulation of CYP2B by IL-1. Treatment of HeLa cells expressing c-Myc-tagged CYP2B1 with NOC-18 down-regulated its expression and enhanced its ubiquitination. Treatment of rat liver microsomes with S-nitrosoglutathione caused S-nitrosylation of CYP2B protein and enhanced the ubiquitination pattern of CYP2B compared with unmodified CYP2B in an in vitro ubiquitination assay. These data are consistent with the hypothesis that NO-dependent CYP2B ubiquitination and proteasomal degradation are dependent on protein modification by reactive nitrogen species.

WITHDRAWN: Endosomal trafficking of EGFR regulated by hVps18 via interaction of MVB sorting machinery.

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Spongiform neurodegeneration-associated E3 ligase Mahogunin ubiquitylates TSG101 and regulates endosomal trafficking.

A null mutation in the gene encoding the putative E3 ubiquitin-protein ligase Mahogunin causes spongiform neurodegeneration, a recessively transmitted prion-like disease in mice. However, no substrates of Mahogunin have been identified, and the cellular role of Mahogunin is unknown. Here, we report the identification of TSG101, a key component of the endosomal sorting complex required for transport (ESCRT)-I, as a specific Mahogunin substrate. We find that Mahogunin interacts with the ubiquitin E2 variant (UEV) domain of TSG101 via its PSAP motif and that it catalyzes monoubiquitylation of TSG101 both in vivo and in vitro. Depletion of Mahogunin by small interfering RNAs in mammalian cells disrupts endosome-to-lysosome trafficking of epidermal growth factor receptor, resulting in prolonged activation of a downstream signaling cascade. Our findings support a role for Mahogunin in a proteasome-independent ubiquitylation pathway and suggest a link between dysregulation of endosomal trafficking and spongiform neurodegeneration.

The interaction of mammalian Class C Vps with nSec-1/Munc18-a and syntaxin 1A regulates pre-synaptic release.

Membrane docking and fusion in neurons is a highly regulated process requiring the participation of a large number of SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and SNARE-interacting proteins. We found that mammalian Class C Vps protein complex associated specifically with nSec-1/Munc18-a, and syntaxin 1A both in vivo and in vitro. In contrast, VAMP2 and SNAP-25, other neuronal core complex proteins, did not interact. When co-transfected with the human growth hormone (hGH) reporter gene, mammalian Class C Vps proteins enhanced Ca2+-dependent exocytosis, which was abolished by the Ca2+-channel blocker nifedipine. In hippocampal primary cultures, the lentivirus-mediated overexpression of hVps18 increased asynchronous spontaneous synaptic release without changing mEPSCs. These results indicate that mammalian Class C Vps proteins are involved in the regulation of membrane docking and fusion through an interaction with neuronal specific SNARE molecules, nSec-1/Munc18-a and syntaxin 1A.

Expression of the mammalian homologue of vacuolar protein sorting 16 (Vps16p) in the mouse and rat brain.

Synaptic vesicle fusion events are essential for synaptic transmission. Membrane docking and fusion events are highly regulated processes requiring the participation of a large number of soluble N-ethylmaleimide-sensitive factors (SNAREs) and SNARE-interacting proteins. We report the neuronal expression of mammalian homologue of vacuolar protein sorting 16 (mVps16p) which exhibits a high homology to the yeast Vps16p, a component of Class C Vps. Western blot and immunohistochemical analyzes revealed that mVps16p is highly expressed in the various brain areas and developmental stages tested. The immunoreactivities of mVps16p colocalized with microtubule associated protein 2, but not glial fibrillary acidic protein. In the primary culture of cortical neurons, mVps16p immunoreactivities were observed in the cell body and the neuronal processes, and highly enriched in the axonal outgrowths.

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes.

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.

Identification of mouse Vps16 and biochemical characterization of mammalian class C Vps complex.

Many multiprotein complexes mediate the fusion of the intracellular membranes. The question how the specificity of the membrane fusion is controlled has not been fully elucidated. Here we report the identification of a mouse homologue Vps16p (mVps16), which exhibits a high homology to the yeast Vps16p, a component of Class C vacuolar protein sorting (Vps) complex implicated in the yeast vacuole membrane fusion. Northern and Western blot analyses reveal that mVps16 is ubiquitously expressed in the mouse peripheral tissues. Biochemical analyses show that mammalian Class C Vps proteins interact with multiple syntaxins and Vps45p, which localizes in the endosomal compartments. The internalization of transferrin (Tf) is not affected by the overexpression of mammalian class C Vps proteins, but the recycling was inhibited. Taken together, this study provides biochemical characteristics of mVps16p in mammalian cells and the potential roles of mammalian Class C Vps proteins in membrane trafficking.