Application of Reducible Covalent Capture Purification for Resolving Persulfidome and Nucleolin S-Sulfhydration.

Protein S-sulfhydration involves the regulation of various protein functions, and resolving the S-sulfhydrated proteome (persulfidome) allows for a deeper exploration of various redox regulations. Therefore, we designed a reducible covalent capture method for isolating S-sulfhydrated proteins, which can analyze the persulfidome in biological samples and monitor specific S-sulfhydrated proteins. In this study, we applied this method to reveal the S-sulfhydration levels of proteins, including 3-phosphoglyceraldehyde dehydrogenase, NFκB/p65, and nucleolin. Furthermore, this technique can be used to enrich S-sulfhydrated peptides, aiding in the determination of protein S-sulfhydration modification sites. Finally, we observed that the S-sulfhydration and oxidation of nucleolin on the C543 residue correlate with its nuclear translocation, downstream regulation of p53, Bcl-xL, and Bcl-2 RNA levels and protein expression, as well as the protective function against oxidative stress. Therefore, this method may facilitate the understanding of the regulation of protein function by redox perturbation.

The study of isotopic enrichment of water in human plasma and erythrocyte.

Life does not sustain without water. For water, there is a natural abundance of stable isotope hydrogen and oxygen. Water molecules get across cell membranes through a plasma membrane protein, named aquaporin. Moreover, the kidney is the main organ to maintain water homeostasis. Here, we study the stable isotopic ratios of hydrogen and oxygen in human blood plasma and erythrocyte corresponding to kidney functions. We extract waters from human plasma and erythrocyte, collected from 110 participants, including 51 clinically stable outpatients with end-stage renal disease (ESRD) and 59 subjects with normal renal function (NRF). We observed that (i) both extracellular (blood plasma) and intracellular (erythrocyte) biology waters are isotopic differences between the ESRD and NRF participants, (ii) the natural abundance of isotopic waters of ESRD is hypo-isotopic, and (iii) the isotopic enrichment of water between erythrocyte and blood plasma are distinct. In addition, we introduce an empirical formula using entropy transformation to describe isotopic water enrichment for biology. Accordingly, the natural abundance of stable isotope water of blood plasma and erythrocyte may be possibly put in practice a new sign for assessments of kidney dysfunctions.

New insights into the regulation of placental growth factor gene expression by the transcription factors GCM1 and DLX3 in human placenta.

Expression of placental growth factor (PGF) is closely associated with placental perfusion in early pregnancy. PGF is primarily expressed in placental trophoblasts, and its expression decreases in preeclampsia, associated with placental hypoxia. The transcription factors glial cells missing 1 (GCM1) and metal-regulatory transcription factor 1 (MTF1) have been implicated in the regulation of PGF gene expression through regulatory elements upstream and downstream of the PGF transcription start site, respectively. Here, we clarified the mechanism underlying placenta-specific PGF expression. We demonstrate that GCM1 up-regulates PGF expression through three downstream GCM1-binding sites (GBSs) but not a previously reported upstream GBS. Interestingly, we also found that these downstream GBSs also harbor metal-response elements for MTF1. Surprisingly, however, we observed that MTF1 is unlikely to regulate PGF expression in the placenta because knockdown or overexpression of GCM1, but not MTF1, dramatically decreased PGF expression or reversed the suppression of PGF expression under hypoxia, respectively. We also demonstrate that another transcription factor, Distal-less homeobox 3 (DLX3), interacts with the DNA-binding domain and the first transactivation domain of GCM1 and that this interaction inhibits GCM1-mediated PGF expression. Moreover, the GCM1-DLX3 interaction interfered with CREB-binding protein-mediated GCM1 acetylation and activation. In summary, we have identified several GBSs in the PGF promoter that are highly responsive to GCM1, have demonstrated that MTF1 does not significantly regulate PGF expression in placental cells, and provide evidence that DLX3 inhibits GCM1-mediated PGF expression. Our findings revise the mechanism for GCM1- and DLX3-mediated regulation of PGF gene expression.

Quantitative display of the redox status of proteins with maleimide-polyethylene glycol tagging.

Cysteine oxidation, either biologically reversible or irreversible, is the main posttranslational modification associated with redox signaling and oxidative stress. Maleimide-polyethylene glycol (m-PEG) has been used to detect reversibly oxidized proteins by reacting to the reduced cysteine residues leading to mobility shift in immunoblots; a method called PEG-switch. With PEG-switch, both reduced and oxidized proteins can be observed on the same immunoblot simultaneously, providing a simple quantitative measurement for protein thiol modifications. In this report, we optimized the assay conditions and exploited the applications of PEG-switch in quantitation of the extent of protein thiol oxidation in cells in response to H2 O2 and insulin. In addition, we have proposed a redox scoring system for measuring the redox status of any given protein from the m-PEG immunoblot. Our results provided quantitative data showing that two cysteine residues of protein tyrosine phosphatase 1B are prone to oxidation following insulin treatment in cultured HeLa cells.

Quantitative Proteomics Analysis Reveals the Min System of Escherichia coli Modulates Reversible Protein Association with the Inner Membrane.

The Min system of Escherichia coli mediates placement of the division septum at the midcell. It oscillates from pole to pole to establish a concentration gradient of the division inhibition that is high at the poles but low at the midcell; the cell middle thereby becomes the most favorable site for division. Although Min oscillation is well studied from molecular and biophysical perspectives, it is still an enigma as to whether such a continuous, energy-consuming, and organized movement of the Min proteins would affect cellular processes other than the division site selection. To tackle this question, we compared the inner membrane proteome of the wild-type and Δmin strains using a quantitative approach. Forty proteins that showed differential abundance on the inner membrane of the mutant cells were identified and defined as proteins of interest (POIs). More than half of the POIs were peripheral membrane proteins, suggesting that the Min system affects mainly reversible protein association with the inner membrane. In addition, 6 out of 10 selected POIs directly interacted with at least one of the Min proteins, confirming the correlation between POIs and the Min system.Further analysis revealed a functional relationship between metabolism and the Min system. Metabolic enzymes accounted for 45% of the POIs, and there was a change of metabolites in the related reactions. We hypothesize that the Min system could alter the membrane location of proteins to modulate their enzymatic activity. Thus, the metabolic modulation in the Δmin mutant is likely an adaptive phenotype in cells of abnormal size and chromosome number due to an imbalanced abundance of proteins on the inner membrane. Taken together, the current work reports novel interactions of the Min system and reveals a global physiological impact of the Min system in addition to the division site placement.

Urinary glycated uromodulin in diabetic kidney disease.

Advanced glycation end-products (AGEs) form during oxidative stress, which is increased in diabetes mellitus (DM). Uromodulin is a protein with a renal protective effect, and may be subject to glycation. The implications of uromodulin glycation and AGEs in the urine are not understood. Here, immunoprecipitation and liquid chromatography-mass spectrometry identified glycated uromodulin (glcUMOD) in the urine of 62.5% of patients with diabetic kidney disease (DKD), 20.0% of patients with non-diabetic chronic kidney disease (CKD), and no DM patients with normal renal function or healthy control participants; a finding replicated in a larger cohort of 84 patients with CKD in a case-control study (35 with DM, 49 without). Uromodulin forms high molecular weight polymers that associate with microvesicles and exosomes. Differential centrifugation identified uromodulin in the supernatant, microvesicles, and exosomes of the urine of healthy participants, but only in the supernatant of samples from patients with DKD, suggesting that glycation influences uromodulin function. Finally, the diagnostic and prognostic utility of measuring urinary glcUMOD concentration was examined. Urinary glcUMOD concentration was substantially higher in DKD patients than non-diabetic CKD patients. Urinary glcUMOD concentration predicted DKD status, particularly in patients with CKD stages 1-3a aged <65 years and with urine glcUMOD concentration ≥9,000 arbitrary units (AU). Urinary uromodulin is apparently glycated in DKD and forms AGEs, and glcUMOD may serve as a biomarker for DKD.

Enrichment of Metabolite-Binding Proteins by Affinity Elution in Tandem Hydrophobic Interaction Chromatography (AETHIC) Reveals RKIP Regulating ERK Signaling in an ATP-Dependent Manner.

To elucidate the molecular mechanisms underlying the action of bioactive compounds such as metabolites, identification of their binding targets is essential. However, available techniques for enriching metabolite-binding proteins are practically restrained by special equipment requirements and laborious efforts. Here we have developed a novel method, affinity elution in tandem hydrophobic interaction chromatography (AETHIC), which enables enrichment of metabolite-binding proteins from a crude tissue extract. AETHIC constitutes two major steps, protein fractionation and affinity elution. The basic strategy of AETHIC uses a series of HIC matrices encompassing aliphatic chains of different length and thus provides a wide range of hydrophobicity for interactions with most proteins. Thereafter, target proteins are eluted selectively by a given ligand. As our first proof-of-principle, we demonstrated that AETHIC was able to enrich ATP-binding proteins from porcine brain extract. In addition, we have demonstrated that raf kinase inhibitory protein (RKIP) is an ATP-binding protein and ATP attenuates the interaction between RKIP and Raf-1. In parallel, short-term ATP depletion in cultured HEK293 cells augments interaction between RKIP and Raf-1, resulting in decreased activation of the downstream ERK signaling. Therefore, the ATP-binding function renders RKIP's inhibition on Raf-1 modulated by cellular ATP concentrations. These data shed light on how energy levels affect the propagation of cellular signaling. Taken together, the enclosed results advocate the potential of AETHIC in the study of metabolite-protein interactions.

White spot syndrome virus protein kinase 1 defeats the host cell's iron-withholding defense mechanism by interacting with host ferritin.

UNLABELLED: Iron is an essential nutrient for nearly all living organisms, including both hosts and invaders. Proteins such as ferritin regulate the iron levels in a cell, and in the event of a pathogenic invasion, the host can use an iron-withholding mechanism to restrict the availability of this essential nutrient to the invading pathogens. However, pathogens use various strategies to overcome this host defense. In this study, we demonstrated that white spot syndrome virus (WSSV) protein kinase 1 (PK1) interacted with shrimp ferritin in the yeast two-hybrid system. A pulldown assay and 27-MHz quartz crystal microbalance (QCM) analysis confirmed the interaction between PK1 and both ferritin and apoferritin. PK1 did not promote the release of iron ions from ferritin, but it prevented apoferritin from binding ferrous ions. When PK1 was overexpressed in Sf9 cells, the cellular labile iron pool (LIP) levels were elevated significantly. Immunoprecipitation and atomic absorption spectrophotometry (AAS) further showed that the number of iron ions bound by ferritin decreased significantly at 24 h post-WSSV infection. Taken together, these results suggest that PK1 prevents apoferritin from iron loading, and thus stabilizes the cellular LIP levels, and that WSSV uses this novel mechanism to counteract the host cell's iron-withholding defense mechanism. IMPORTANCE: We show here that white spot syndrome virus (WSSV) ensures the availability of iron by using a previously unreported mechanism to defeat the host cell's iron-withholding defense mechanism. This defense is often implemented by ferritin, which can bind up to 4,500 iron atoms and acts to sequester free iron within the cell. WSSV's novel counterstrategy is mediated by a direct protein-protein interaction between viral protein kinase 1 (PK1) and host ferritin. PK1 interacts with both ferritin and apoferritin, suppresses apoferritin's ability to sequester free iron ions, and maintains the intracellular labile iron pool (LIP), and thus the availability of free iron is increased within cells.

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 virus's requirements for energy and macromolecular precursors. The WSSV-induced Warburg effect therefore appears to be essential for successful viral replication.

Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis.

The polyamines spermidine and spermine, and their precursor putrescine, have been shown to play an important role in cell migration, proliferation, and differentiation. Because of their polycationic property, polyamines are traditionally thought to be involved in DNA replication, gene expression, and protein translation. However, polyamines can also be covalently conjugated to proteins by transglutaminase 2 (TG2). This modification leads to an increase in positive charge in the polyamine-incorporated region which significantly alters the structure of proteins. It is anticipated that protein polyamine conjugation may affect the protein-protein interaction, protein localization, and protein function of the TG2 substrates. In order to investigate the roles of polyamine modification, we synthesized a spermine-conjugated antigen and generated an antiserum against spermine. In vitro TG2-catalyzed spermine incorporation assays were carried out to show that actin, tubulins, heat shock protein 70 and five types of histone proteins were modified with spermine, and modification sites were also identified by liquid chromatography and linear ion trap-orbitrap hybrid mass spectrometry. Subsequent mass spectrometry-based shotgun proteomic analysis also identified 254 polyaminated sites in 233 proteins from the HeLa cell lysate catalyzed by human TG2 with spermine, thus allowing, for the first time, a global appraisal of site-specific protein polyamination. Global analysis of mouse tissues showed that this modification really exists in vivo. Importantly, we have demonstrated that there is a new histone modification, polyamination, in cells. However, the functional significance of histone polyamination demands further investigations.

The genome and occlusion bodies of marine Penaeus monodon nudivirus (PmNV, also known as MBV and PemoNPV) suggest that it should be assigned to a new nudivirus genus that is distinct from the terrestrial nudiviruses.

BACKGROUND: Penaeus monodon nudivirus (PmNV) is the causative agent of spherical baculovirosis in shrimp (Penaeus monodon). This disease causes significant mortalities at the larval stage and early postlarval (PL) stage and may suppress growth and reduce survival and production in aquaculture. The nomenclature and classification status of PmNV has been changed several times due to morphological observation and phylogenetic analysis of its partial genome sequence. In this study, we therefore completed the genome sequence and constructed phylogenetic trees to clarify PmNV's taxonomic position. To better understand the characteristics of the occlusion bodies formed by this marine occluded virus, we also compared the chemical properties of the polyhedrin produced by PmNV and the baculovirus AcMNPV (Autographa californica nucleopolyhedrovirus). RESULTS: We used next generation sequencing and traditional PCR methods to obtain the complete PmNV genome sequence of 119,638 bp encoding 115 putative ORFs. Phylogenetic tree analysis showed that several PmNV genes and sequences clustered with the non-occluded nudiviruses and not with the baculoviruses. We also investigated the characteristics of PmNV polyhedrin, which is a functionally important protein and the major component of the viral OBs (occlusion bodies). We found that both recombinant PmNV polyhedrin and wild-type PmNV OBs were sensitive to acid conditions, but unlike the baculoviral OBs, they were not susceptible to alkali treatment. CONCLUSIONS: From the viral genome features and phylogenetic analysis we conclude that PmNV is not a baculovirus, and that it should be assigned to the proposed Nudiviridae family with the other nudiviruses, but into a distinct new genus (Gammanudivirus).

Zebrafish Dkk3a protein regulates the activity of myf5 promoter through interaction with membrane receptor integrin α6b.

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane receptors of Dkk3a, and Integrin α6b (Itgα6b) was finally identified. To further confirm this, we used cell surface binding assays, which showed that Dkk3a and Itgα6b were co-expressed at the cell membrane of HEK-293T cells. Cross-linking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the ß-propeller repeat domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was up-regulated 4-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a knockdown embryos co-injected with itgα6b mRNA and itgα6b knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to that in control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker or absent myf5 expression, and reduced the protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.

Involvement of Epac1/Rap1/CaMKI/HDAC5 signaling cascade in the regulation of placental cell fusion.

The placental transcription factor glial cell missing 1 (GCM1) and its target gene syncytin-1 are involved in cAMP-stimulated trophoblastic fusion for syncytiotrophoblast formation. GCM1 DNA-binding activity is inhibited by sumoylation, whereas GCM1 stability is decreased by deacetylation. cAMP enhances GCM1 desumoylation through the Epac1/Rap1/CaMKI signaling cascade and CaMKI is known to down-regulate class IIa HDAC activity. In this paper, we study whether the Epac1/Rap1/CaMKI signaling cascade regulates GCM1 activity and placental cell fusion through class IIa HDACs. Interaction and co-localization of GCM1 and HDAC5 were characterized by co-immunoprecipitation analysis and immunofluorescence microscopy (IFM). Regulation of GCM1 transcription activity and syncytin-1 expression by HDAC5 was studied by transient expression. Phospho-specific antibodies against HDAC5, RNA interference and IFM were used to examine the de-repression of GCM1 activity, syncytin-1 expression and cell-cell fusion by Epac1/Rap1/CaMKI signaling cascade in placental BeWo cells expressing constitutively active Epac1 and CaMKI. We demonstrate that both GCM1 and HDAC5 are expressed in the syncytiotrophoblast layer of full-term placenta and the nuclei of BeWo cells. The interaction between HDAC5 and GCM1 facilitates GCM1 deacetylation and suppresses its transcriptional activity. In contrast, Epac1 stimulates HDAC5 phosphorylation on Ser259 and Ser498 in a Rap1- and CaMKI-dependent manner leading to nuclear export of HDAC5 and thereby de-repression of GCM1 transcriptional activity. Importantly, HDAC5 suppresses syncytin-1 expression and cell-cell fusion in BeWo cells, which is counteracted by Epac1 and CaMKI. Our results reveal a new layer of regulation of GCM1 activity and placental cell fusion through the Epac1/Rap1/CaMKI signaling cascade by restraining HDAC5 from interacting with and mediating GCM1 deacetylation.

Dual-specificity phosphatase 23 mediates GCM1 dephosphorylation and activation.

Glial cells missing homolog 1 (GCM1) is a transcription factor essential for placental development. GCM1 promotes syncytiotrophoblast formation and placental vasculogenesis by activating fusogenic and proangiogenic gene expression in placenta. GCM1 activity is regulated by multiple post-translational modifications. The cAMP/PKA-signaling pathway promotes CBP-mediated GCM1 acetylation and stabilizes GCM1, whereas hypoxia-induced GSK-3ß-mediated phosphorylation of Ser322 causes GCM1 ubiquitination and degradation. How and whether complex modifications of GCM1 are coordinated is not known. Here we show that the interaction of GCM1 and dual-specificity phosphatase 23 (DUSP23) is enhanced by PKA-dependent phosphorylation of GCM1 on Ser269 and Ser275. The recruitment of DUSP23 reverses GSK-3ß-mediated Ser322 phosphorylation, which in turn promotes GCM1 acetylation, stabilization and activation. Supporting a central role in coordinating GCM1 modifications, knockdown of DUSP23 suppressed GCM1 target gene expression and placental cell fusion. Our study identifies DUSP23 as a novel factor that promotes placental cell fusion and reveals a complex regulation of GCM1 activity by coordinated phosphorylation, dephosphorylation and acetylation.

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.

Proteomic identification of a novel hsp90-containing protein-mineral complex which can be induced in cells in response to massive calcium influx.

Fetuin-A is known for limiting the expansion and formation of hydroxyapatite crystals from calcium phosphate aggregates in circulation by forming a soluble fetuin-mineral complex. This study was aimed to uncover potential proteins involved in the regulation of calcium phosphate precipitation within cells. We found that a novel protein-mineral complex (PMC) can be generated after introduction of calcium chloride and sodium phosphate into the porcine brain protein extract prepared in Tris-HCl buffer. Selectively enriched proteins in the pellet were confirmed by immunoblotting, including heat shock protein 90 (Hsp90), annexin A5, calreticulin, nucleolin, and other proteins. In addition, purified native Hsp90 directly bound both amorphous calcium phosphate and hydroxyapatite and underwent conformational changes and oligomerization in the presence of excess calcium and phosphate. The morphology of the PMC prepared from Hsp90, calcium, and phosphate was distinctly different from that of hydroxyapatite under transmission electron microscope observation. When cultured SiHa cells were treated with a calcium ionophore or damaged by scratch to induce the massive calcium influx, a complex was formed and observed at discrete sites near the plasma membrane as revealed by antibodies against Hsp90, annexin A5, calreticulin, nucleolin, and other proteins. This complex could also be probed in situ with fetuin-A suggesting the existence of calcium phosphate aggregates in this complex. Inhibition of the complex formation by bisphosphonates hindered cell recovery from A23187 assault. Our results show that following membrane damage amorphous calcium phosphate develops at sites near membrane rupture where saturated calcium phosphate concentration is achieved. As a result, Hsp90 and other proteins are recruited, and the cytosolic PMC is formed. Inhibition of the cytosolic PMC formation may in part contribute to the cellular toxicity and in vivo side effects of bisphosphonates, particularly in cells prone to membrane damage under physiological conditions such as gastrointestinal epithelial and oral cavity epithelial cells.

White spot syndrome virus induces metabolic changes resembling the warburg effect in shrimp hemocytes in the early stage of infection.

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.

Functional antagonism between ΔNp63α and GCM1 regulates human trophoblast stemness and differentiation.

The combination of EGF, CHIR99021, A83-01, SB431542, VPA, and Y27632 (EGF/CASVY) facilitates the derivation of trophoblast stem (TS) cells from human blastocysts and first-trimester, but not term, cytotrophoblasts. The mechanism underlying this chemical induction of TS cells remains elusive. Here we demonstrate that the induction efficiency of cytotrophoblast is determined by functional antagonism of the placental transcription factor GCM1 and the stemness regulator ΔNp63α. ΔNp63α reduces GCM1 transcriptional activity, whereas GCM1 inhibits ΔNp63α oligomerization and autoregulation. EGF/CASVY cocktail activates ΔNp63α, thereby partially inhibiting GCM1 activity and reverting term cytotrophoblasts into stem cells. By applying hypoxia condition, we can further reduce GCM1 activity and successfully induce term cytotrophoblasts into TS cells. Consequently, we identify mitochondrial creatine kinase 1 (CKMT1) as a key GCM1 target crucial for syncytiotrophoblast differentiation and reveal decreased CKMT1 expression in preeclampsia. Our study delineates the molecular underpinnings of trophoblast stemness and differentiation and an efficient method to establish TS cells from term placentas.

Potential role of follicle-stimulating hormone (FSH) and transforming growth factor (TGFß1) in the regulation of ovarian angiogenesis.

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)ß1 is known to facilitate FSH-induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFß1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin-primed immature rats and treated once with FSH and/or TGFß1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFß1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFß1 > FSH ≈ TGFß1 >control) that was partly attributed to the increased secretion of pro-angiogenic factors VEGF and PDGF-B. This is further supported by the evidence that pre-treatment with inhibitor of VEGF receptor-2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2-day aortic ring culture period suppressed microvessel growth in GCCM-treated groups, and also inhibited the FSH + TGFß1-GCCM-stimulated release of matrix remodeling-associated gelatinase activities. Interestingly, pre-treatment of AG1296 at late stage suppressed GCCM-induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFß1 increased the secretion of VEGF and PDGF-B, and that in turn up-regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFß1 play important roles in regulation of ovarian angiogenesis during follicle development.

Role of tissue transglutaminase 2 in the acquisition of a mesenchymal-like phenotype in highly invasive A431 tumor cells.

BACKGROUND: Cancer progression is closely linked to the epithelial-mesenchymal transition (EMT) process. Studies have shown that there is increased expression of tissue tranglutaminase (TG2) in advanced invasive cancer cells. TG2 catalyzes the covalent cross-linking of proteins, exhibits G protein activity, and has been implicated in the modulation of cell adhesion, migration, invasion and cancer metastasis. This study explores the molecular mechanisms associated with TG2's involvement in the acquisition of the mesenchymal phenotype using the highly invasive A431-III subline and its parental A431-P cells. RESULTS: The A431-III tumor subline displays increased expression of TG2. This is accompanied by enhanced expression of the mesenchymal phenotype, and this expression is reversed by knockdown of endogenous TG2. Consistent with this, overexpression of TG2 in A431-P cells advanced the EMT process. Furthermore, TG2 induced the PI3K/Akt activation and GSK3ß inactivation in A431 tumor cells and this increased Snail and MMP-9 expression resulting in higher cell motility. TG2 also upregulated NF-κB activity, which also enhanced Snail and MMP-9 expression resulting in greater cell motility; interestingly, this was associated with the formation of a TG2/NF-κB complex. TG2 facilitated acquisition of a mesenchymal phenotype, which was reversed by inhibitors of PI3K, GSK3 and NF-κB. CONCLUSIONS: This study reveals that TG2 acts, at least in part, through activation of the PI3K/Akt and NF-κB signaling systems, which then induce the key mediators Snail and MMP-9 that facilitate the attainment of a mesenchymal phenotype. These findings support the possibility that TG2 is a promising target for cancer therapy.