Biological Roles of the Podospora anserina Mitochondrial Lon Protease and the Importance of Its N-Domain

Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction

Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli

Membrane interaction of off-pathway prion oligomers and lipid-induced on-pathway intermediates during prion conversion: A clue for neurotoxicity

International audienceSoluble oligomers of prion proteins (PrP), produced during amyloid aggregation, have emerged as the primary neurotoxic species, instead of the fibrillar end-products, in transmissible spongiform encephalopathies. However, whether the membrane is among their direct targets, that mediate the downstream adverse effects, remains a question of debate. Recently, questions arise from the formation of membrane-active oligomeric species generated during the β-aggregation pathway, either in solution, or in lipid environment. In the present study, we characterized membrane interaction of off-pathway oligomers from recombinant prion protein generated along the amyloid aggregation and compared to lipid-induced intermediates produced during lipid-accelerated fi-brillation. Using calcein-leakage assay, we show that the soluble prion oligomers are the most potent in producing leakage with negatively charged vesicles. Binding affinities, conformational states, mode of action of the different PrP assemblies were determined by thioflavin T binding-static light scattering experiments on DOPC/ DOPS vesicles, as well as by FTIR-ATR spectroscopy and specular neutron reflectivity onto the corresponding supported lipid bilayers. Our results indicate that the off-pathway PrP oligomers interact with lipid membrane via a distinct mechanism, compared to the inserted lipid-induced intermediates. Thus, separate neurotoxic mechanisms could exist following the puzzling intermediates generated in the different cell compartments. These results not only reveal an important regulation of lipid membrane on PrP behavior but may also provide clues for designing stage-specific and prion-targeted therapy

A survey of the anesthetic management of pediatric kidney transplantation in France

International audienceBACKGROUND:Renal transplantation is the best available therapeutic option for end-stage renal failure in both children and adults. However, little is known about anesthetic practice during pediatric renal transplantation.MATERIAL AND METHODS:The study consisted of a national survey about anesthetic practice during pediatric renal transplantation in France. French tertiary pediatric centers performing renal transplants were targeted, and one physician from each team was asked to complete the survey. The survey included patient data, preoperative assessment and optimization data, and intraoperative anesthesia data (drugs, ventilation, and hemodynamic interventions).RESULTS:Twenty centers performing kidney transplantation were identified and contacted to complete the survey, and eight responded. Surveyed centers performed 96 of the 122 pediatric kidney transplantations performed in France in 2017 (79%). Centers consistently performed echocardiography and ultrasound examinations of the great veins preoperatively and consistently employed esophageal Doppler cardiac output estimation and vasopressors intraoperatively. All other practices were found to be heterogeneous. Central venous pressure was monitored in six centers, and dopamine was administered perioperatively in two centers.CONCLUSIONS:The current study provides a snapshot of the perioperative management of pediatric kidney transplantation in France. Results emphasize the need for both standardization of practice and awareness of recent evidence against the use of CVP monitoring and dopamine infusions

BRCA1-Dependent Translational Regulation in Breast Cancer Cells

<div><p>BRCA1 (Breast Cancer 1) has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair and protein ubiquitination. We previously demonstrated that BRCA1 interacts with PABP1 (Poly(A)-Binding Protein 1) and that BRCA1 modulates protein synthesis through this interaction. To identify the mRNAs that are translationally regulated by BRCA1, we used a microarray analysis of polysome-bound mRNAs in BRCA1-depleted and non-depleted MCF7 cells. Our findings show that BRCA1 modifies the translational efficiency of approximately 7% of the mRNAs expressed in these cells. Further analysis revealed that several processes contributing to cell surveillance such as cell cycle arrest, cell death, cellular growth and proliferation, DNA repair and gene expression, are largely enriched for the mRNAs whose translation is impacted by BRCA1. The BRCA1-dependent translation of these species of mRNAs therefore uncovers a novel mechanism through which BRCA1 exerts its onco-suppressive role. In addition, the BRCA1-dependent translation of mRNAs participating in unexpected functions such as cellular movement, nucleic acid metabolism or protein trafficking is indicative of novel functions for BRCA1. Finally, this study contributes to the identification of several markers associated with BRCA1 deficiency and to the discovery of new potential anti-neoplastic therapeutic targets.</p></div

BRCA1 is a ribosome-associated protein.

<p>A/MCF7 cells were lysed in 25 mM KCl buffer and the post-mitochondrial cytoplasmic lysate was layered onto a 1 M sucrose cushion and centrifuged as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067313#s2" target="_blank">Material and methods</a>” section. Immunoblotting for BRCA1 using the MS110 antibody was performed on the following samples: initial total cell lysate (L), nuclear fraction (N), cytoplasmic fraction (C) and ribosome pellet (R). PABP1 and eIF4G were used as markers for pellet fraction containing ribosome-associated proteins. The analyzed L, N and C fractions represent 5% of the total cell lysate. B/MCF7 cells were lysed in 25 mM KCl buffer and the cytoplasmic fraction was separated onto a 10–40% sucrose gradient. (Top) A characteristic ribosome profile. (Middle) Extracts of total RNA from half of each fraction were subjected to gel analysis to determine the presence of 18S and 28S rRNAs. rRNAs were detected by Gel Red staining. (Bottom) The remaining half of each fraction was precipitated with TCA. BRCA1 protein was identified with immunoblot analysis using D9 antibody. PABP1 and eIF4G served as controls.</p

Microarray analysis of polysome-associated RNAs from MCF7 cells in which BRCA1 has been depleted.

<p>A/Western blot confirming siRNA inhibition of BRCA1 levels in MCF7 cells when compared with control siRNA. Immunoblotting for BRCA1 used 8F7 antibody. α-tubulin served as loading control. B/Number of mRNAs exhibiting altered translational efficiency in BRCA1-depleted MCF7 cells compared to control MCF7 cells. The 1151 mRNAs displaying a modified relative translatability (RR = polyRNA/totRNA) were clustered in several groups depending on their fold change in polysomal RNA abundance (PolyRNA) and their fold change in total mRNA abundance (TotRNA). The fold changes in polysomal RNA abundance and in total mRNA abundance are indicated as follows: ≤0.67, (↗)≥1.50, (↔) >0.67 and <1.50. The RRs are annotated with a sign and a number. The sign specifies the RR value: (−) ≤0.67, (+) ≥1.50. The number indicates how many mRNAs are deregulated. : mRNAs translationally deregulated through change in polysome mRNA abundance only; : mRNAs translationally deregulated through change in total mRNA abundance only; : mRNAs translationally deregulated through change in polysome abundance together with opposite changes in total mRNA C/Functional distribution of differentially translated known genes in BRCA1-depleted versus control MCF7 cells. Gene functions were established based on the annotation provided by the IPA database. The number of genes enriched in each function is shown in brackets.</p

RT-qPCR analyses of differentially translated mRNAs upon BRCA1 depletion.

<p>Total RNA and polysomal-associated RNA from MCF7 cells transfected with BRCA1-targetting siRNA or control siRNA were reverse transcribed and five transcripts identified in the microarray analysis were quantified by real time PCR. qPCR analysis was performed in triplicate. Analysis of mRNA levels for each target was normalized to HPRT1 mRNA. For each gene, the polyRNA (grey) and the totalRNA (black) Ratios were determined using the ΔΔCt calculation method. Results are representative of the average RNA ratio ± SEM from four independent experiments. *, <i>p</i><0.05 compared with SiControl.</p

Heavy-ion test of detectors with conventional and resistive Micromegas used in TPC configuration

International audienceWe have performed tests of Micromegas detector prototypes using the heavy-ion beams from the SIS synchrotron of GSI (Darmstadt, Germany). The beams varied from (12)C(6+) to (179)Au(65+) and from 250 to 1000 MeV per nucleon. We have tested two amplification technologies, conventional and resistive Micromegas, and two construction concepts, bulk-Micromegas and micro-meshes screwed on the PCB. The obtained position resolution below 200 mu m for 5 mm wide strips implies that the bulk resistive Micromegas technology might meet the requirements of the future R3B TPC project. We also developed a fast and very low noise front-end electronics connected directly to the Printed Circuit Board (PCB) of the detector itself. This concept has shown very good performances and robustness

Interplay of the Serine/Threonine-Kinase StkP and the Paralogs DivIVA and GpsB in Pneumococcal Cell Elongation and Division

International audienc