Geen-Dong Chang

GCMa Regulates the Syncytin-mediated Trophoblastic Fusion

The human placental trophoblast cell can be classified as either a cytotrophoblast or a syncytiotrophoblast. Cytotrophoblasts can function as stem cells for the development of the syncytiotrophoblast layer via cell fusion. An envelope gene of the human endogenous retrovirus family W (HERV-W) calledsyncytin is specifically expressed in the syncytiotrophoblast layer. Syncytin is a fusogenic membrane protein; therefore, it can mediate the fusion of cytotrophoblasts into the syncytiotrophoblast layer, which is essential for pregnancy maintenance. GCMa is a placenta-specific transcription factor and is required for placental development. To study the placenta-specific fusion mediated by syncytin, we tested whether GCMa is involved in this process by regulating syncytin gene expression. In this report, we demonstrate that GCMa was able to regulatesyncytin gene expression via two GCMa-binding sites upstream of the 5′-long terminal repeat of thesyncytin-harboring HERV-W family member in BeWo and JEG3 cells but not in HeLa cells. Furthermore, adenovirus-directed expression of GCMa enhanced syncytin gene expression and syncytin-mediated cell fusion in BeWo and JEG3 cells but not in HeLa cells. Therefore, the integration site of thesyncytin-harboring HERV-W family member in the human genome is close to the functional GCMa-binding sites by which GCMa can specifically transactivate syncytin gene expression in trophoblast cells. Our results may help to explain the mechanism underlying the cell fusion event specific for syncytiotrophoblast formation.

Functional Characterization of the Placental Fusogenic Membrane Protein Syncytin

Abstract Fusion of cytotrophoblasts into the multinucleated syncytiotrophoblast layer is essential for the development of a functional placenta. The envelope protein of a human endogenous retrovirus W (HERV-W) family member, syncytin 1, has been shown to mediate placental cell fusion. Recently, the envelope protein of another HERV family member (HERV-FRD), syncytin 2, has been identified and shown to be highly expressed in the placenta. To better understand the biology of syncytin 2, in this study we first investigated syncytin 2 gene expression in normal and preeclamptic placentas and then characterized the functions of syncytin 2. The expression of syncytin 2 gene was decreased in preeclamptic placentas and could be stimulated by the cAMP stimulant forskolin. The endoprotease furin was found to be involved in the posttranslational cleavage of syncytin 1 and 2 polypeptides into surface and transmembrane subunits. In addition, proper association of the subunits of syncytins 1 and 2 is probably required for the functional integrity of each protein, because subunit swapping of syncytins 1 and 2 failed to generate fusogenic chimeras. Finally, we demonstrated that the disulfide bridge-forming CX2C and CX7C motifs found in syncytins 1 and 2 are essential for their fusogenic activities, because mutations in the CX2C motif not only abolished fusogenesis but also functioned as dominant-negative mutants. Our results suggest that syncytin 2 may function as a second fusogenic protein for placental cell fusion..

White Spot Syndrome Virus Induces Metabolic Changes Resembling the Warburg Effect in Shrimp Hemocytes in the Early Stage of Infection

ABSTRACT The Warburg effect is an abnormal glycolysis response that is associated with cancer cells. Here we present evidence that metabolic changes resembling the Warburg effect are induced by a nonmammalian virus. When shrimp were infected with white spot syndrome virus (WSSV), changes were induced in several metabolic pathways related to the mitochondria. At the viral genome replication stage (12 h postinfection [hpi]), glucose consumption and plasma lactate concentration were both increased in WSSV-infected shrimp, and the key enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PDH), showed increased activity. We also found that at 12 hpi there was no alteration in the ADP/ATP ratio and that oxidative stress was lower than that in uninfected controls. All of these results are characteristic of the Warburg effect as it is present in mammals. There was also a significant decrease in triglyceride concentration starting at 12 hpi. At the late stage of the infection cycle (24 hpi), hemocytes of WSSV-infected shrimp showed several changes associated with cell death. These included the induction of mitochondrial membrane permeabilization (MMP), increased oxidative stress, decreased glucose consumption, and disrupted energy production. A previous study showed that WSSV infection led to upregulation of the voltage-dependent anion channel (VDAC), which is known to be involved in both the Warburg effect and MMP. Here we show that double-stranded RNA (dsRNA) silencing of the VDAC reduces WSSV-induced mortality and virion copy number. For these results, we hypothesize a model depicting the metabolic changes in host cells at the early and late stages of WSSV infection.

Endocytic pathway is required for Drosophila Toll innate immune signaling

The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. Here we show that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. We conclude that endocytosis is required for the activation of the Toll signaling pathway.

Histone deacetylase 3 binds to and regulates the GCMa transcription factor

Human GCMa transcription factor regulates expression of syncytin, a placental fusogenic protein mediating trophoblastic fusion. Recently, we have demonstrated that CBP-mediated GCMa acetylation underlies the activated cAMP/PKA signaling pathway that stimulates trophoblastic fusion. Because protein acetylation is a reversible modification governed by histone acetyltransferases (HATs) and histone deacetylase (HDACs), in this study we investigated the key HDACs responsible for deacetylation of GCMa and thus the reduction in GCMa activity to avoid unwanted fusion events that may have adverse effects on placental morphogenesis. We herein demonstrate that the HDAC inhibitor, trichostatin A (TSA), increases the level of acetylated GCMa and that HDAC1, 3, 4 and 5 interact with and deacetylate GCMa. Glutathione S-transferase (GST) pull-down assays further verified direct interaction between GCMa and HDAC3 or CBP and HDAC3. HDAC3 counteracts the transcriptional coactivator activity of CBP and the enhancement effect of CBP on GCMa-mediated transcriptional activation. Correlatively, we found in placental cells that HDAC3 associates with the proximal GCMa-binding site (pGBS) in the syncytin promoter and dissociates from pGBS in the presence of forskolin, which stimulates the association of CBP and GCMa with pGBS. Our studies support that trophoblastic fusion in placental morphogenesis depends on the regulation of GCMa activity by HAT and HDAC.

Improvement of glycosylation in insect cells with mammalian glycosyltransferases

The N-glycans of recombinant glycoproteins expressed in insect cells mainly contain high mannose or tri-mannose structures, which are truncated forms of the sialylated N-glycans found in mammalian cells. Because asialylated glycoproteins have a shorter half-life in blood circulation, we investigated if sialylated therapeutic glycoprotein can be produced from insect cells by enhancing the N-glycosylation machinery of the cells. We co-expressed in two insect cell lines, Sf9 and Ea4, the human alpha1-antitrypsin (halpha1AT) protein with a series of key glycosyltransferases, including GlcNAc transferase II (GnT2), beta1,4-galactosyltransferase (beta14GT), and alpha2,6-sialyltransferase (alpha26ST) by a single recombinant baculovirus. We demonstrated that the enhancement of N-glycosylation is cell type-dependent and is more efficient in Ea4 than Sf9 cells. Glycan analysis indicated that sialylated halpha1AT proteins were produced in Ea4 insect cells expressing the above-mentioned exogenous glycosyltransferases. Therefore, our expression strategy may simplify the production of humanized therapeutic glycoproteins by improving the N-glycosylation pathway in specific insect cells, with an ensemble of exogenous glycosyltransferases in a single recombinant baculovirus.

An Invertebrate Warburg Effect: A Shrimp Virus Achieves Successful Replication by Altering the Host Metabolome via the PI3K-Akt-mTOR Pathway

In this study, we used a systems biology approach to investigate changes in the proteome and metabolome of shrimp hemocytes infected by the invertebrate virus WSSV (white spot syndrome virus) at the viral genome replication stage (12 hpi) and the late stage (24 hpi). At 12 hpi, but not at 24 hpi, there was significant up-regulation of the markers of several metabolic pathways associated with the vertebrate Warburg effect (or aerobic glycolysis), including glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis. We show that the PI3K-Akt-mTOR pathway was of central importance in triggering this WSSV-induced Warburg effect. Although dsRNA silencing of the mTORC1 activator Rheb had only a relatively minor impact on WSSV replication, in vivo chemical inhibition of Akt, mTORC1 and mTORC2 suppressed the WSSV-induced Warburg effect and reduced both WSSV gene expression and viral genome replication. When the Warburg effect was suppressed by pretreatment with the mTOR inhibitor Torin 1, even the subsequent up-regulation of the TCA cycle was insufficient to satisfy the viruss requirements for energy and macromolecular precursors. The WSSV-induced Warburg effect therefore appears to be essential for successful viral replication.

FBW2 Targets GCMa to the Ubiquitin-Proteasome Degradation System

The GCM proteins GCMa/1 and GCMb/2 are novel zinc-containing transcription factors critical for glial cell differentiation in fly and for placental as well as parathyroid gland development in mouse. Previous pulse-chase experiments have demonstrated differential protein stabilities of GCM proteins with half-lives from ∼30 min to 2 h (Tuerk, E. E., Schreiber, J., and Wegner, M. (2000) J. Biol. Chem. 275, 4774–4782). However, little is known about the machinery that controls GCM protein degradation. Here, we report the identification of an SCF complex as the GCM ubiquitin-protein isopeptide ligase (E3) that regulates human GCMa (hGCMa) degradation. We found that SKP1 and CUL1, two key components of the SCF complex, associate with hGCMa in vivo. We further identify the human F-box protein FBW2 (hFBW2) as the substrate recognition subunit in the SCF E3 complex for hGCMa. We show that hFBW2 interacts with hGCMa in a phosphorylation-dependent manner and promotes hGCMa ubiquitination. Supporting a critical role for hFBW2 in hGCMa degradation, knockdown of hFBW2 expression by RNA interference leads to a reduction in hGCMa ubiquitination and a concomitant increase in hGCMa protein stability. Our study identifies the SCFhFBW2 E3 complex as the key machinery that targets hGCMa to the ubiquitin-proteasome degradation system.

Purification and molecular cloning of carp ovarian cystatin

The ovarian fluid of carp consists of many components. Using the antiserum against carp serum, Western blot analysis of ovarian fluid was done in order to distinguish substances synthesized by the ovary from those derived from the serum. Several ovary-specific substances were detected including a protein of 12 kDa (p12), which was purified to homogeneity. Purified p12 displays a single band in SDS-PAGE under nonreducing condition and it can inhibit the enzymatic activity of papain with an apparent inhibition constant of 0.01 nM. The primary structure of p12 was partially determined by Edman degradation and fully elucidated by molecular cloning. A cDNA of 531 bp encoding p12 was obtained. The precursor of p12 has 129 residues, including a signal peptide of 18 residues and a mature protein of 111 residues. The N- and C-terminus of p12 are threonine and methionine, respectively. The p12 shares many common features of the family 2 cystatins of other species, including the similarity of the protein size (in the range of 110 to 120 residues), the presence of 4 cysteine residues and the occurrence of invariant residues throughout the molecule.

A novel cyclic AMP/Epac1/CaMKI signaling cascade promotes GCM1 desumoylation and placental cell fusion.

ABSTRACT Cyclic AMP (cAMP) signaling and the placental transcription factor glial cell missing 1 (GCM1) regulate expression of syncytin-1 and -2 fusogenic proteins, which are critical for syncytiotrophoblast formation by trophoblast fusion. We recently revealed a cAMP/protein kinase A (PKA)/CBP signaling pathway that activates GCM1 by coordinating GCM1 phosphorylation and acetylation. In contrast, GCM1 activity is downregulated by sumoylation of Lys156. How GCM1 sumoylation is regulated was unknown. Here, we identify a novel PKA-independent cAMP signaling pathway as the critical regulator of GCM1 sumoylation. We show that Epac1 and Rap1, in response to cAMP, activate CaMKI to phosphorylate Ser47 in GCM1. This phosphorylation facilitates the interaction between GCM1 and the desumoylating enzyme SENP1 and thereby leads to GCM1 desumoylation and activation. Using RNA interference (RNAi), we further demonstrate that 8-(4-chlorophenylthio)-2′-O-Me-cAMP-AM (8-CPT-AM), an Epac activator, stimulates syncytin-1 and -2 gene expression and cell fusion of placental BeWo cells in a GCM1-dependent manner. Importantly, the cell fusion defect in GCM1-knockdown BeWo cells can be reversed and enhanced by the RNAi-resistant phosphomimetic GCM1(S47D) mutant. Our study has identified a novel cAMP/Epac1/CaMKI/GCM1 signaling cascade that stimulates trophoblast fusion through promoting GCM1 phosphorylation and desumoylation.