The role of ERp44 in maturation of serotonin transporter protein

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.     

Studies on serum gamma-glutamyl transpeptidase in primary idiopathic hypothyroidism patients.

Variations in the levels of serum gamma-glutamyl transpeptidase (GGT) were measured in control subjects and in 39 adult primary idiopathic hypothyroidism (PIH) patients. The serum GGT activity was low in PIH patients compared to that of control subjects. A more significant correlation was found between serum GGT and T3 (r = 0.766) but not with T4 (r = 0.476). The comparison of serum GGT with TSH has revealed that those two parameters are not parallel with each other (r = -0.454). No significant correlation between serum GGT activity, age, and sex in PIH patients and control subjects was observed. The present available data indicate that measurement of serum GGT might be useful as a marker index in PIH patients.     

Inhibition of Thromboxane Synthase Activity Improves Glioblastoma Response to Alkylation Chemotherapy

Thromboxane synthase (TXSA), an enzyme of the arachidonic acid metabolism, is upregulated in human glial tumors and is involved in glioma progression. Here, we analyzed the in vitro and in vivo effects of pharmacological inhibition of TXSA activity on human glioblastoma cells. Furegrelate, a specific inhibitor of TXSA, significantly inhibited tumor growth in an orthotopic glioblastoma model by inducing proapoptotic, antiproliferative, and antiangiogenic effects. Inhibition of TXSA induced a proapoptotic disposition of glioma cells and increased the sensitivity to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea, significantly prolonging the survival time of intracerebral glioma-bearing mice. Our data demonstrate that the targeted inhibition of TXSA activity improves the efficiency of conventional alkylation chemotherapy in vivo. Our study supports the role of TXSA activity for the progression of malignant glioma and the potential utility of its therapeutic modulation for glioma treatment.     

Temporal differences in microRNA expression patterns in astrocytes and neurons after ischemic injury

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that modulate gene translation. Their expression is altered in many central nervous system (CNS) injuries suggesting a role in the cellular response to stress. Current studies in brain tissue have not yet described the cell-specific temporal miRNA expression patterns following ischemic injury. In this study, we analyzed the expression alterations of a set of miRNAs in neurons and astrocytes subjected to 60 minutes of ischemia and collected at different time-points following this injury. To mimic ischemic conditions and reperfusion in vitro, cortical primary neuronal and astrocytic cultures prepared from fetal rats were first placed in oxygen and glucose deprived (OGD) medium for 60 minutes, followed by their transfer into normoxic pre-conditioned medium. Total RNA was extracted at different time-points after the termination of the ischemic insult and the expression levels of miRNAs were measured. In neurons exposed to OGD, expression of miR-29b was upregulated 2-fold within 6 h and up to 4-fold at 24 h post-OGD, whereas induction of miR-21 was upregulated 2-fold after 24 h when compared to expression in neurons under normoxic conditions. In contrast, in astrocytes, miR-29b and miR-21 were upregulated only after 12 h. MiR-30b, 107, and 137 showed expression alteration in astrocytes, but not in neurons. Furthermore, we show that expression of miR-29b was significantly decreased in neurons exposed to Insulin-Like Growth Factor I (IGF-I), a well documented neuroprotectant in ischemic models. Our study indicates that miRNAs expression is altered in neurons and astrocytes after ischemic injury. Furthermore, we found that following OGD, specific miRNAs have unique cell-specific temporal expression patterns in CNS. Therefore the specific role of each miRNA in different intracellular processes in ischemic brain and the relevance of their temporal and spatial expression patterns warrant further investigation that may lead to novel strategies for therapeutic interventions.     

The Cellular Distribution of Serotonin Transporter Is Impeded on Serotonin-Altered Vimentin Network

Background

The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments.

Methodology/Principal Findings

We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin.To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Δ14 and Δ20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S₆₁₁, T₆₁₃, and T₆₁₆ arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter.

Conclusions/Significance

Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network.     

Towards guidance values for the environmental performance of buildings: application to the statistical analysis of 40 low-energy single family houses’ LCA in France

Purpose

In this study, life cycle assessment (LCA) is applied to a sample of 40 low-energy individual houses for the French context in order to identify guidance values for different environmental priorities (energy and water consumption, greenhouse gases emissions, waste generation etc.).

Methods

Calculation rules for the LCA derived from EeBGuide guidance and HQE Performance specific rules for the French context. Data are based on Environmental Product Declaration (EPD for the impacts related to products and technical equipment while generic data are used for energy and water processes. The LCA is defined for the entire life cycle of a building from cradle-to-grave according to NF EN 15978 standard. It includes the products and equipment implemented in the building, the different uses of energy for heating, domestic hot water, lighting, ventilation and auxiliaries, and the different uses of water consumption.

Results and discussion

Results for the 40 houses showed that the average life cycle non-renewable primary energy consumption is about 37 kWh/(m²*year) while the life cycle greenhouse gases emissions are of 8.4 kg CO₂-eq/(m²*year). The embodied impacts represent between 40% and 72% for the following indicators: acidification, global warming, non-renewable primary energy, and radioactive waste. The net fresh water use is mostly determined by the direct use of the water in use, and the non-hazardous waste indicator is only linked to the materials and equipment. When integrating the variability of the different houses design, energy performance, climate requirements, it was found that those values can vary of an order of two between the 10 and 90% percentiles’ values. It was found that the results are also sensitive to the enlargement of the system boundaries (e.g. inclusion of the other uses of energy such as building appliances) and the modification of the reference study period.

Conclusions and recommendations

This study provided a first set of LCA guidance values describing a range of environmental impacts for new low-energy individual houses in France. Results were also reported for different design parameters, system boundaries and reference study period. The outcomes of this study can now serve as a basis to guide and support new LCA-based labelling systems developed by public authorities and labelling schemes (e.g. the HQE Association).

     

Cis regulation within a cluster of viral microRNAs

MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi''s sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the pre-miR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.     

Impact of Elevated Plasma Serotonin on Global Gene Expression of Murine Megakaryocytes

Background

Serotonin (5-HT) is a biogenic amine that also acts as a mitogen and a developmental signal early in rodent embryogenesis. Genetic and pharmacological disruption of 5-HT signaling causes various diseases and disorders via mediating central nervous system, cardiovascular system, and serious abnormalities on a growing embryo. Today, neither the effective modulators on 5-HT signaling pathways nor the genes affected by 5-HT signal are well known yet.

Methodology/Principal Findings

In an attempt to identify the genes altered by 5-HT signaling pathways, we analyzed the global gene expression via the Illumina array platform using the mouse WG-6 v2.0 Expression BeadChip containing 45,281 probe sets representing 30,854 genes in megakaryocytes isolated from mice infused with 5-HT or saline. We identified 723 differentially expressed genes of which 706 were induced and 17 were repressed by elevated plasma 5-HT.

Conclusions/Significance

Hierarchical gene clustering analysis was utilized to represent relations between groups and clusters. Using gene ontology mining tools and canonical pathway analyses, we identified multiple biological pathways that are regulated by 5-HT: (i) cytoskeletal remodeling, (ii) G-protein signaling, (iii) vesicular transport, and (iv) apoptosis and survival. Our data encompass the first extensive genome-wide based profiling in the progenitors of platelets in response to 5-HT elevation in vivo.