Cross-Talk between Oxysterols and Glucocorticoids: Differential Regulation of Secreted Phopholipase A2 and Impact on Oligodendrocyte Death

BACKGROUND: Oxysterols are oxidized forms of cholesterol. They have been shown to be implicated in cholesterol turnover, inflammation and in neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis. Glial cells are targets of oxysterols: they inhibit astrocyte proliferation after brain injury, and we have previously shown that 25-hydroxycholesterol (25OH) provokes oligodendrocyte apoptosis and stimulates the expression of sPLA2 type IIA (sPLA2-IIA), which has a protective effect. METHODOLOGY/PRINCIPAL FINDINGS: As glucocorticoids are well-known for their anti-inflammatory effects, our aim was to understand their direct effects on oxysterol-induced responses in oligodendrocytes (sPLA2-IIA stimulation and apoptosis). We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) abolishes the stimulation of sPLA2-IIA by 25-hydroxycholesterol (25-OH). This inhibition is mediated by the glucocorticoid receptor (GR), which decreases the expression of the oxysterol receptor Pregnane X Receptor (PXR) and interferes with oxysterol signaling by recruiting a common limiting coactivator PGC1alpha. Consistent with the finding that sPLA2-IIA can partially protect oligodendrocytes against oxysterol-triggered apoptosis, we demonstrate here that the inhibition of sPLA2-IIA by Dex accelerates the apoptotic phenomenon, leading to a shift towards necrosis. We have shown by atomic force microscopy and electron microscopy that 25-OH and Dex alters oligodendrocyte shape and disorganizes the cytoplasm. CONCLUSIONS/SIGNIFICANCE: Our results provide a new understanding of the cross-talk between oxysterol and glucocorticoid signaling pathways and their respective roles in apoptosis and oligodendrocyte functions

Nuclear Glycolytic Enzyme Enolase of Toxoplasma Gondii Functions as a Transcriptional Regulator

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii

Characterization of a nuclear pore protein sheds light on the roles and composition of the Toxoplasma gondii nuclear pore complex

International audienceThe nuclear pore is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Here, we report the characterization of the first Toxoplasma gondii nuclear pore protein, named TgNup302, which appears to be the orthologue of the mammalian Nup98-96 protein. We produced a conditional knock-down mutant that expresses TgNup302 under the control of an inducible tetracycline-regulated promoter. Under ATc treatment, a substantial decrease of TgNup302 protein in inducible knock-down (iKD) parasites was observed, causing a delay in parasite proliferation. Moreover, the nuclear protein TgENO2 was trapped in the cytoplasm of ATc-treated mutants, suggesting that TgNup302 is involved in nuclear transport. Fluorescence in situ hybridization revealed that TgNup302 is essential for 18S RNA export from the nucleus to the cytoplasm, while global mRNA export remains unchanged. Using an affinity tag purification combined with mass spectrometry, we identified additional components of the nuclear pore complex, including proteins potentially interacting with chromatin. Furthermore, reverse immunoprecipitation confirmed their interaction with TgNup302, and structured illuminated microscopy confirmed the NPC localization of some of the TgNup302-interacting proteins. Intriguingly, facilitates chromatin transcription complex (FACT) components were identified, suggesting the existence of an NPC-chromatin interaction in T. gondii. Identification of TgNup302-interacting proteins also provides the first glimpse at the NPC structure in Apicomplexa, suggesting a structural conservation of the NPC components between distant eukaryotes

A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

International audienceIn Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii

Remodeling of Channel-Forming ORAI Proteins Determines an Oncogenic Switch in Prostate Cancer

SummaryORAI family channels have emerged as important players in malignant transformation, yet the way in which they reprogram cancer cells remains elusive. Here we show that the relative expression levels of ORAI proteins in prostate cancer are different from that in noncancerous tissue. By mimicking ORAI protein remodeling observed in primary tumors, we demonstrate in in vitro models that enhanced ORAI3 expression favors heteromerization with ORAI1 to form a novel channel. These channels support store-independent Ca2+ entry, thereby promoting cell proliferation and a smaller number of functional homomeric ORAI1-based store-operated channels, which are important in supporting susceptibility to apoptosis. Thus, our findings highlight disrupted dynamic equilibrium of channel-forming proteins as an oncogenic mechanism

Visualizing the 3D Architecture of Multiple Erythrocytes Infected with Plasmodium at Nanoscale by Focused Ion Beam-Scanning Electron Microscopy

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures

Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion

Toxoplasma (toxoplasmosis) and Plasmodium (malaria) use unique secretory organelles for migration, cell invasion, manipulation of host cell functions, and cell egress. In particular, the apical secretory micronemes and rhoptries of apicomplexan parasites are essential for successful host infection. New findings reveal that the contents of these organelles, which are transported through the endoplasmic reticulum (ER) and Golgi, also require the parasite endosome-like system to access their respective organelles. In this review, we discuss recent findings that demonstrate that these parasites reduced their endosomal system and modified classical regulators of this pathway for the biogenesis of apical organelles

Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF)

Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells and cancer-associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis

TLR3 modulates immunopathology during a Schistosoma mansoni egg-driven Th2 response in the lung

We examined the role of TLR3 in Th2-driven pulmonary granulomatous disease, using wildtype (TLR3 +/+ ) and TLR3 gene-deficient (TLR3 −/− ) mice in a well-established model of Schistosoma mansoni egg-induced pulmonary granuloma. The intravenous bolus injection of S. mansoni eggs into S. mansoni -sensitized TLR3 +/+ mice was associated with an increase in TLR3 transcript expression in alveolar macrophages and ex vivo spleen and lung cultures at day 8 after egg injection. Lungs from TLR3 −/− mice showed an increase in granuloma size, greater collagen deposition around the granuloma, and increased Th2 cytokine and chemokine levels compared with similarly sensitized and challenged TLR3 +/+ mice. Macrophages from TLR3 −/− mice exhibited an M2 phenotype characterized by increased arginase and CCL2 expression. Significantly greater numbers of CD4 + CD25 + T cells were present in the lungs of TLR3 −/− mice compared with TLR3 +/+ mice at day 8 after egg embolization. Cells derived from granulomatous lung and lung draining lymph nodes of TLR3 −/− mice released significantly higher levels of IL-17 levels relative to TLR3 +/+ cells. Thus, our data suggest that TLR3 has a major regulatory role during a Th2-driven granulomatous response as its absence enhanced immunopathology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61443/1/3436_ftp.pd

Formation of the Food Vacuole in Plasmodium falciparum: A Potential Role for the 19 kDa Fragment of Merozoite Surface Protein 1 (MSP119)

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase