Publications by authors named "Johana Chicher"

31 Publications

The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis.

Nat Commun 2021 02 26;12(1):1298. Epub 2021 Feb 26.

Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, Strasbourg, France.

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3' terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.
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http://dx.doi.org/10.1038/s41467-021-21382-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910438PMC
February 2021

Proteasome subunit PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress.

EMBO Mol Med 2020 07 5;12(7):e11861. Epub 2020 Jun 5.

Laboratoire de Génétique Médicale, INSERM, UMRS_1112, Institut de Génétique Médicale d'Alsace (IGMA), Université de Strasbourg, Faculté de médecine de Strasbourg, Strasbourg, France.

The ubiquitin-proteasome system degrades ubiquitin-modified proteins to maintain protein homeostasis and to control signalling. Whole-genome sequencing of patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient's fibroblasts was however unaffected. Nevertheless, patient's cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient's fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient's cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development.
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http://dx.doi.org/10.15252/emmm.201911861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338805PMC
July 2020

The Arabidopsis mTERF-repeat MDA1 protein plays a dual function in transcription and stabilization of specific chloroplast transcripts within the psbE and ndhH operons.

New Phytol 2020 09 26;227(5):1376-1391. Epub 2020 May 26.

Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France.

The mTERF gene family encodes for nucleic acid binding proteins that are predicted to regulate organellar gene expression in eukaryotes. Despite the implication of this gene family in plant development and response to abiotic stresses, a precise molecular function was assigned to only a handful number of its c. 30 members in plants. Using a reverse genetics approach in Arabidopsis thaliana and combining molecular and biochemical techniques, we revealed new functions for the chloroplast mTERF protein, MDA1. We demonstrated that MDA1 associates in vivo with components of the plastid-encoded RNA polymerase and transcriptional active chromosome complexes. MDA1 protein binds in vivo and in vitro with specificity to 27-bp DNA sequences near the 5'-end of psbE and ndhA chloroplast genes to stimulate their transcription, and additionally promotes the stabilization of the 5'-ends of processed psbE and ndhA messenger (m)RNAs. Finally, we provided evidence that MDA1 function in gene transcription likely coordinates RNA folding and the action of chloroplast RNA-binding proteins on mRNA stabilization. Our results provide examples for the unexpected implication of DNA binding proteins and gene transcription in the regulation of mRNA stability in chloroplasts, blurring the boundaries between DNA and RNA metabolism in this organelle.
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http://dx.doi.org/10.1111/nph.16625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496394PMC
September 2020

The nature of the purine at position 34 in tRNAs of 4-codon boxes is correlated with nucleotides at positions 32 and 38 to maintain decoding fidelity.

Nucleic Acids Res 2020 06;48(11):6170-6183

Institut de Biologie Moléculaire et Cellulaire, 'Architecture et Réactivité de l'ARN' CNRS UPR9002, Université de Strasbourg, 2, allée Konrad Roentgen, F-67084 Strasbourg, France.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.
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http://dx.doi.org/10.1093/nar/gkaa221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293025PMC
June 2020

YBEY is an essential biogenesis factor for mitochondrial ribosomes.

Nucleic Acids Res 2020 09;48(17):9762-9786

UMR7156 - Molecular Genetics, Genomics, Microbiology, University of Strasbourg, CNRS, Strasbourg F-67000, France.

Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.
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http://dx.doi.org/10.1093/nar/gkaa148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515705PMC
September 2020

Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication-Associated Proteins.

Proteomics 2020 02 13;20(3-4):e1900184. Epub 2020 Feb 13.

Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, UMR7242, CNRS, Illkirch, 67412, France.

It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA-PK complex (DNA-PKcs and the Ku heterodimer) recognizes, and is activated by, structured single-stranded DNA is provided. Hijacking the DNA-PK complex by double-stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA-PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non-Homologous End-Joining (NHEJ) pathway, especially the XRCC4-DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA-PK complex interferes with replication fork dynamics in vivo.
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http://dx.doi.org/10.1002/pmic.201900184DOI Listing
February 2020

RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation.

Nucleic Acids Res 2019 10;47(18):9871-9887

CIRI, Centre International de Recherche en Infectiologie, Inserm U1111, Université Lyon1, Ecole Normale Supérieure de Lyon, CNRS UMR5308, Lyon, France.

The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5' end while its 3' part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, sodA mRNA was identified as a primary target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3'UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. The mature and stable form of RsaC inhibits the synthesis of the Mn-containing enzyme SodA synthesis and favors the oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. In addition, other putative targets of RsaC are involved in oxidative stress (ROS and NOS) and metal homeostasis (Fe and Zn). Consequently, RsaC may balance two interconnected defensive responses, i.e. oxidative stress and metal-dependent nutritional immunity.
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http://dx.doi.org/10.1093/nar/gkz728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765141PMC
October 2019

RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis.

Nat Commun 2019 08 27;10(1):3871. Epub 2019 Aug 27.

Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, Strasbourg, France.

The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.
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http://dx.doi.org/10.1038/s41467-019-11807-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711988PMC
August 2019

Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not.

PLoS One 2019 28;14(3):e0214313. Epub 2019 Mar 28.

INRA, UMR1331, Toxalim, Research Centre in Food Toxicology, Toulouse, France.

The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays-the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214313PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438463PMC
December 2019

Small is big in Arabidopsis mitochondrial ribosome.

Nat Plants 2019 01 9;5(1):106-117. Epub 2019 Jan 9.

Institut de biologie de moléculaire des plantes UPR2357 du CNRS, Université de Strasbourg, Strasbourg, France.

Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.
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http://dx.doi.org/10.1038/s41477-018-0339-yDOI Listing
January 2019

ATG5 is required for B cell polarization and presentation of particulate antigens.

Autophagy 2019 02 22;15(2):280-294. Epub 2018 Sep 22.

a CNRS, Immunology, Immunopathology and Therapeutic Chemistry , Institut de Biologie Moléculaire et Cellulaire/University of Strasbourg , Strasbourg , France.

The involvement of macroautophagy/autophagy proteins in B-cell receptor (BCR) trafficking, although suspected, is not well understood. We show that ATG5 (autophagy related 5) contributes to BCR polarization after stimulation and internalization into LAMP1 (lysosomal-associated membrane protein 1) and major histocompatibility complex class II (MHC-II) compartments. BCR polarization is crucial in the context of immobilized antigen processing. Moreover, antigen presentation to cognate T cells is decreased in the absence of ATG5 when the model antigen OVAL/ovalbumin is provided in an immobilized form in contrast to the normal presentation of soluble OVAL. We further show that ATG5 is required for centrosome polarization and actin nucleation in the immune synapse area. This event is accompanied by an increased interaction between ATG16L1 (autophagy related 16-like 1 []) and the microtubule-organizing center-associated protein PCM1 (pericentriolar material 1). In the human B cell line BJAB, PCM1 is required for BCR polarization after stimulation. We thus propose that the ATG12 (autophagy related 12)-ATG5-ATG16L1 complex under BCR stimulation allows its interaction with PCM1 and consequently facilitates centrosome relocalization to the immune synapse, optimizing the presentation of particulate antigens. ACTB: actin beta; ACTR2/3: ARP2/3 actin-related protein 2/3; APC: antigen-presenting cells; ATG: autophagy-related; BCR: B cell receptor; BECN1/Beclin 1: beclin 1, autophagy related; CDC42: cell division cycle 42; Cr2: complement receptor 2; CSFE: carboxyfluorescein succinimidyl ester; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; EEA1: early endosome antigen 1; ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothyocyanate; GC: germinal center; GJA1/CX3: gap junction protein, alpha 1; Ig: immunoglobulin; LAMP1: lysosomal-associated membrane protein 1; LAP: LC3-associated phagocytosis; LM: littermate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen activated protein kinase; MHC-II: major histocompatibility complex class II; MIIC: MHC class II compartment; OVAL: ovalbumin; PBS: phosphate-buffered saline; PCM1: pericentriolar material 1; PtdIns3K: phosphatidylinositol 3-kinase; PTPRC/CD45RB/B220; Protein tyrosine phosphatase, receptor type, C; SYK: spleen tyrosine kinase; TBS: Tris-buffered saline; TCR: T cell receptor; ULK1: unc-51 like kinase 1.
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http://dx.doi.org/10.1080/15548627.2018.1516327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333460PMC
February 2019

The UPF1 interactome reveals interaction networks between RNA degradation and translation repression factors in Arabidopsis.

Plant J 2018 10 1;96(1):119-132. Epub 2018 Aug 1.

Institut de biologie moléculaire des plantes (IBMP), CNRS, Université de Strasbourg, 67000, Strasbourg, France.

The RNA helicase UP-FRAMESHIFT (UPF1) is a key factor of nonsense-mediated decay (NMD), a mRNA decay pathway involved in RNA quality control and in the fine-tuning of gene expression. UPF1 recruits UPF2 and UPF3 to constitute the NMD core complex, which is conserved across eukaryotes. No other components of UPF1-containing ribonucleoproteins (RNPs) are known in plants, despite its key role in regulating gene expression. Here, we report the identification of a large set of proteins that co-purify with the Arabidopsis UPF1, either in an RNA-dependent or RNA-independent manner. We found that like UPF1, several of its co-purifying proteins have a dual localization in the cytosol and in P-bodies, which are dynamic structures formed by the condensation of translationally repressed mRNPs. Interestingly, more than half of the proteins of the UPF1 interactome also co-purify with DCP5, a conserved translation repressor also involved in P-body formation. We identified a terminal nucleotidyltransferase, ribonucleases and several RNA helicases among the most significantly enriched proteins co-purifying with both UPF1 and DCP5. Among these, RNA helicases are the homologs of DDX6/Dhh1, known as translation repressors in humans and yeast, respectively. Overall, this study reports a large set of proteins associated with the Arabidopsis UPF1 and DCP5, two components of P-bodies, and reveals an extensive interaction network between RNA degradation and translation repression factors. Using this resource, we identified five hitherto unknown components of P-bodies in plants, pointing out the value of this dataset for the identification of proteins potentially involved in translation repression and/or RNA degradation.
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http://dx.doi.org/10.1111/tpj.14022DOI Listing
October 2018

Toxicological effects of CdSe nanocrystals on the marine diatom Phaeodactylum tricornutum: The first mass spectrometry-based proteomic approach.

Ecotoxicol Environ Saf 2018 May 4;152:78-90. Epub 2018 Feb 4.

Institut National des Sciences et Techniques de la Mer, Conservatoire National des Arts et Métiers, 50103 Cherbourg en Cotentin Cedex, France; Laboratoire Universitaire des Sciences Appliquées de Cherbourg, EA4253, Normandie Université, UNICAEN, 50130 Cherbourg en Cotentin, France. Electronic address:

In the marine environment, benthic diatoms from estuarine and coastal sediments are among the first targets of nanoparticle pollution whose potential toxicity on marine organisms is still largely unknown. It is therefore relevant to improve our knowledge of interactions between these new pollutants and microalgae, the key players in the control of marine resources. In this study, the response of P. tricornutum to CdSe nanocrystals (CdSe NPs) of 5 nm (NP5) and 12 nm (NP12) in diameter was evaluated through microscopic, physiological, biochemical and proteomic approaches. NP5 and NP12 affected cell growth but oxygen production was only slightly decreased by NP5 after 1-d incubation time. In our experimental conditions, a high CdSe NP dissolution was observed during the first day of culture, leading to Cd bioaccumulation and oxidative stress, particularly with NP12. However, after a 7-day incubation time, proteomic analysis highlighted that P. tricornutum responded to CdSe NP toxicity by regulating numerous proteins involved in protection against oxidative stress, cellular redox homeostasis, Ca regulation and signalling, S-nitrosylation and S-glutathionylation processes and cell damage repair. These proteome changes allowed algae cells to regulate their intracellular ROS level in contaminated cultures. P. tricornutum was also capable to control its intracellular Cd concentration at a sufficiently low level to preserve its growth. To our knowledge, this is the first work allowing the identification of proteins differentially expressed by P. tricornutum subjected to NPs and thus the understanding of some molecular pathways involved in its cellular response to nanoparticles.

Significance: The microalgae play a key role in the control of marine resources. Moreover, they produce 50% of the atmospheric oxygen. CdSe NPs are extensively used in the industry of renewable energies and it is regrettably expected that these pollutants will sometime soon appear in the marine environment through surface runoff, urban effluents and rivers. Since estuarine and coastal sediments concentrate pollutants, benthic microalgae which live in superficial sediments will be among the first targets of nanoparticle pollution. Thus, it is relevant to improve our knowledge of interactions between diatoms and nanoparticles. Proteomics is a powerful tool for understanding the molecular mechanisms triggered by nanoparticle exposure, and our study is the first one to use this tool to identify proteins differentially expressed by P. tricornutum subjected to CdSe nanocrystals. This work is fundamental to improve our knowledge about the defence mechanisms developed by algae cells to counteract damage caused by CdSe NPs.
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http://dx.doi.org/10.1016/j.ecoenv.2018.01.043DOI Listing
May 2018

The cryo-EM Structure of a Novel 40S Kinetoplastid-Specific Ribosomal Protein.

Structure 2017 12 26;25(12):1785-1794.e3. Epub 2017 Oct 26.

Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, 67000 Strasbourg, France. Electronic address:

Kinetoplastids are potentially lethal protozoan pathogens affecting more than 20 million people worldwide. There is a critical need for more specific targets for the development of safer anti-kinetoplastid therapeutic molecules that can replace the scarce and highly cytotoxic current drugs. The kinetoplastid ribosome represents a potential therapeutic target due to its relative structural divergence when compared with its human counterpart. However, several kinetoplastid-specific ribosomal features remain uncharacterized. Here, we present the near-atomic cryoelectron microscopy structure of a novel bona fide kinetoplastid-specific ribosomal (r-) protein (KSRP) bound to the ribosome. KSRP is an essential protein located at the solvent face of the 40S subunit, where it binds and stabilizes kinetoplastid-specific domains of rRNA, suggesting its role in ribosome integrity. KSRP also interacts with the r-protein eS6 at a region that is only conserved in kinetoplastids. The kinetoplastid-specific ribosomal environment of KSRP provides a promising target for the design of safer anti-kinetoplastidian drugs.
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http://dx.doi.org/10.1016/j.str.2017.09.014DOI Listing
December 2017

Definition of a RACK1 Interaction Network in Using SWATH-MS.

G3 (Bethesda) 2017 07 5;7(7):2249-2258. Epub 2017 Jul 5.

RIDI UPR 9022, Université de Strasbourg, Centre National de la Recherche Scientifique, F-67000, France

Receptor for Activated protein C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism , we recently showed that RACK1 is required at the ribosome for internal ribosome entry site (IRES)-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition (DDA and DIA, respectively) and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method, both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation, and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus (CrPV), an IRES-containing virus.
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http://dx.doi.org/10.1534/g3.117.042564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499132PMC
July 2017

The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms.

Nucleic Acids Res 2017 Jun;45(11):6746-6760

Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, F-67000 Strasbourg, France.

The virulon of Staphyloccocus aureus is controlled by intricate connections between transcriptional and post-transcriptional regulators including proteins and small non-coding RNAs (sRNAs). Many of the sRNAs regulate gene expression through base-pairings with mRNAs. However, characterization of the direct sRNA targets in Gram-positive bacteria remained a difficult challenge. Here, we have applied and adapted the MS2-affinity purification approach coupled to RNA sequencing (MAPS) to determine the targetome of RsaA sRNA of S. aureus, known to repress the synthesis of the transcriptional regulator MgrA. Several mRNAs were enriched with RsaA expanding its regulatory network. Besides mgrA, several of these mRNAs encode a family of SsaA-like enzymes involved in peptidoglycan metabolism and the secreted anti-inflammatory FLIPr protein. Using a combination of in vivo and in vitro approaches, these mRNAs were validated as direct RsaA targets. Quantitative differential proteomics of wild-type and mutant strains corroborated the MAPS results. Additionally, it revealed that RsaA indirectly activated the synthesis of surface proteins supporting previous data that RsaA stimulated biofilm formation and favoured chronic infections. All together, this study shows that MAPS could also be easily applied in Gram-positive bacteria for identification of sRNA targetome.
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http://dx.doi.org/10.1093/nar/gkx219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499838PMC
June 2017

GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin.

EMBO J 2017 04 28;36(7):886-903. Epub 2017 Feb 28.

Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, UPR 2357, Université de Strasbourg, Strasbourg, France

Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF-containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA-ROP2), contain high levels of active TOR ROP2 physically interacts with and, when GTP-bound, activates TOR TOR activation in response to auxin is abolished in ROP-deficient plants. GFP-TOR can associate with endosome-like structures in ROP2-overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA-ROP2 is efficient in loading uORF-containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD-8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin.
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http://dx.doi.org/10.15252/embj.201694816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376970PMC
April 2017

Structure of the 70S ribosome from human pathogen Staphylococcus aureus.

Nucleic Acids Res 2017 01 28;45(2):1026. Epub 2016 Nov 28.

Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg 67084, France

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http://dx.doi.org/10.1093/nar/gkw1126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314762PMC
January 2017

Structure of the 70S ribosome from human pathogen Staphylococcus aureus.

Nucleic Acids Res 2016 12 18;44(21):10491-10504. Epub 2016 Oct 18.

Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg 67084, France

Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen.
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http://dx.doi.org/10.1093/nar/gkw933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137454PMC
December 2016

A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi.

Nat Commun 2016 11 24;7:13586. Epub 2016 Nov 24.

Medical Genetics Laboratory, INSERM U1112, Institute of Medical Genetics of Alsace, University of Strasbourg, Strasbourg Medical School, 67000 Strasbourg, France.

Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium.
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http://dx.doi.org/10.1038/ncomms13586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123056PMC
November 2016

The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

Front Plant Sci 2016 7;7:1611. Epub 2016 Nov 7.

Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay Versailles, France.

Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.
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http://dx.doi.org/10.3389/fpls.2016.01611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100631PMC
November 2016

Ability of the marine bacterium Pseudomonas fluorescens BA3SM1 to counteract the toxicity of CdSe nanoparticles.

J Proteomics 2016 10 11;148:213-27. Epub 2016 Aug 11.

Institut National des Sciences et Techniques de la Mer, Conservatoire National des Arts et Métiers, 50103 Cherbourg-Octeville Cedex, France.

Unlabelled: In the marine environment, bacteria from estuarine and coastal sediments are among the first targets of nanoparticle pollution; it is therefore relevant to improve the knowledge of interactions between bacteria and nanoparticles. In this work, the response of the marine bacterium Pseudomonas fluorescens BA3SM1 to CdSe nanocrystals (CdSe NPs) of 3nm (NP3) and 8nm (NP8) in diameter was evaluated through microscopic, physiological, biochemical and proteomic approaches. Transmission electron microscopy images showed that NP3 were able to penetrate the bacteria, while NP8 were highly concentrated around the cells, embedded in large exopolysaccharides. In our experimental conditions, both CdSe NP sizes induced a decrease in respiration during the stationary growth phase, while only NP8 caused growth retardation and a decrease in pyoverdine production. Proteomic analyses highlighted that the strain responded to CdSe NP toxicity by inducing various defence mechanisms such as cell aggregation, extracellular CdSe NP sequestration, effective protection against oxidative stress, modifications of envelope organization and properties, and cadmium export. In addition, BA3SM1 presented a biosorption capacity of 1.6×10(16)NP3/g dry weight and 1.7×10(15)NP8/g dry weight. This strain therefore appears as a promising agent for NP bioremediation processes. Proteomic data are available via ProteomeXchange with identifier PXD004012.

Biological Significance: To the best of our knowledge, this is the first report focussing on the effects of CdSe colloidal nanocrystals (CdSe NPs) on a marine strain of Pseudomonas fluorescens. CdSe NPs are extensively used in the industry of renewable energies and it is regrettably expected that these pollutants will sometime soon appear in the marine environment through surface runoff, urban effluents and rivers. Bacteria living in estuarine and coastal sediments will be among the first targets of these new pollutants. The pseudomonads are frequently found in these ecosystems. They are involved in several biogeochemical cycles and are known for their high resistance to pollutants. Consequently, this study focussing on the effects of CdSe NPs on the marine strain P. fluorescens BA3SM1 is highly relevant for several reasons. First, it aims at improving knowledge about the interactions between bacteria and NPs. This is fundamental to effectively use NPs against pathogenic bacteria. Secondly, in spite of CdSe NP interactions with the bacterial cells, the strain BA3SM1 can develop various strategies to counteract CdSe NP toxicity and ensure its growth. It exhibits interesting properties to sequester CdSe NPs and it retains its ability to form biofilm. The strain therefore appears as a promising agent for NP bioremediation thanks to biofiltration processes. Finally, this study shows that CdSe NPs of 8nm in diameter cause a decrease in the secretion of siderophore pyoverdine, a secondary metabolite playing a key role in microbial ecology since it drives bacterial survival and competitiveness in ecosystems. Bacteria producing effective siderophores survive better in a Fe-deficient environment where they antagonize the growth of other microbes thought iron deprivation. Furthermore, siderophores are also employed as virulence factors in human pathogenic strains such as P. aeruginosa. Consequently, this study highlights that NPs can impact the secondary metabolism of bacteria with environmental and medical implications. In addition, in this work, Data-Dependant Acquisition (DDA) provided state of the art Mass Spectrometry data by Spectral Counting and MS1 Label-Free. The combination of these two well-known proteomic techniques including manual validations strengthened the identification and quantification of regulated proteins. Moreover, numerous correlations between proteomic analyses and other observations (physiological, biochemical, microscopic) consolidated our interpretations.
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http://dx.doi.org/10.1016/j.jprot.2016.07.021DOI Listing
October 2016

Proteome analysis of digestive fluids in Nepenthes pitchers.

Ann Bot 2016 Mar;117(3):479-95

INRA UMR 1121, Laboratoire Agronomie et Environnement, 2 avenue de la forêt de Haye TSA 40602, F-54518 Vandœuvre-lès-Nancy, France, Université de Lorraine UMR 1121, Laboratoire Agronomie et Environnement, 2 avenue de la forêt de Haye TSA 40602, F-54518 Vandœuvre-lès-Nancy, France.

Background And Aims: Carnivorous plants have developed strategies to enable growth in nutrient-poor soils. For the genus Nepenthes, this strategy represents producing pitcher-modified leaves that can trap and digest various prey. These pitchers produce a digestive fluid composed of proteins, including hydrolytic enzymes. The focus of this study was on the identification of these proteins.

Methods: In order to better characterize and have an overview of these proteins, digestive fluid was sampled from pitchers at different stages of maturity from five species of Nepenthes (N. mirabilis, N. alata, N. sanguinea, N. bicalcarata and N. albomarginata) that vary in their ecological niches and grew under different conditions. Three complementary approaches based on transcriptomic resources, mass spectrometry and in silico analysis were used.

Key Results: This study permitted the identification of 29 proteins excreted in the pitchers. Twenty of these proteins were never reported in Nepenthes previously and included serine carboxypeptidases, α- and β-galactosidases, lipid transfer proteins and esterases/lipases. These 20 proteins display sequence signals allowing their secretion into the pitcher fluid.

Conclusions: Nepenthes pitcher plants have evolved an arsenal of enzymes to digest prey caught in their traps. The panel of new proteins identified in this study provides new insights into the digestive process of these carnivorous plants.
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http://dx.doi.org/10.1093/aob/mcw001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765550PMC
March 2016

Improvement of Mitochondria Extract from Saccharomyces cerevisiae Characterization in Shotgun Proteomics Using Sheathless Capillary Electrophoresis Coupled to Tandem Mass Spectrometry.

J Chromatogr Sci 2016 Apr 8;54(4):653-63. Epub 2016 Feb 8.

Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, 67008 Strasbourg, France.

In this work, we describe the characterization of a quantity-limited sample (100 ng) of yeast mitochondria by shotgun bottom-up proteomics. Sample characterization was carried out by sheathless capillary electrophoresis, equipped with a high sensitivity porous tip and coupled to tandem mass spectrometry (CESI-MS-MS) and concomitantly with a state-of-art nano flow liquid chromatography coupled to a similar mass spectrometry (MS) system (nanoLC-MS-MS). With single injections, both nanoLC-MS-MS and CESI-MS-MS 60 min-long separation experiments allowed us to identify 271 proteins (976 unique peptides) and 300 proteins (1,765 unique peptides) respectively, demonstrating a significant specificity and complementarity in identification depending on the physicochemical separation employed. Such complementary, maximizing the number of analytes detected, presents a powerful tool to deepen a biological sample's proteomic characterization. A comprehensive study of the specificity provided by each separating technique was also performed using the different properties of the identified peptides: molecular weight, mass-to-charge ratio (m/z), isoelectric point (pI), sequence coverage or MS-MS spectral quality enabled to determine the contribution of each separation. For example, CESI-MS-MS enables to identify larger peptides and eases the detection of those having extreme pI without impairing spectral quality. The addition of peptides, and therefore proteins identified by both techniques allowed us to increase significantly the sequence coverages and then the confidence of characterization. In this study, we also demonstrated that the two yeast enolase isoenzymes were both characterized in the CESI-MS-MS data set. The observation of discriminant proteotypic peptides is facilitated when a high number of precursors with high-quality MS-MS spectra are generated.
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http://dx.doi.org/10.1093/chromsci/bmw005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4885408PMC
April 2016

Identification of factors involved in target RNA-directed microRNA degradation.

Nucleic Acids Res 2016 Apr 24;44(6):2873-87. Epub 2016 Jan 24.

Architecture and Reactivity of RNA, Institut de biologie moléculaire et cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, 67084 Strasbourg, France

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3'-5' exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.
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http://dx.doi.org/10.1093/nar/gkw040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824107PMC
April 2016

Purification of mRNA-programmed translation initiation complexes suitable for mass spectrometry analysis.

Proteomics 2015 Jul 11;15(14):2417-25. Epub 2015 Jun 11.

Institut de Biologie Moléculaire et Cellulaire, "Architecture et Réactivité de l'ARN", Université De Strasbourg, Strasbourg, France.

Liquid Chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) is a powerful analytical technique for the identification and mass analysis of complex protein mixtures. Here, we present a combination of methods developed for the extensive/deep proteomic analysis of purified ribosome/mRNA particles assembled in rabbit reticulocyte lysate (RRL). Ribosomes are assembled on chimeric biotinylated mRNA-DNA molecules immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After washing steps, the complexes are trypsin-digested directly on the beads in semi-native condition or after their elution from the beads in denaturing Laemmli buffer. The nanoLC-MS/MS analysis performed on complexes assembled on β-globin, viral HCV, and histone H4 mRNAs revealed significant differences in initiation factors composition in agreement with models of translation initiation used by these different types of mRNAs. Using Laemmli-denaturing condition induces release of deeply buried peptides from the ribosome and eukaryotic initiation factor 3 (eIF3) allowing the identification of the nearly complete set of ribosomal proteins.
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http://dx.doi.org/10.1002/pmic.201400628DOI Listing
July 2015

Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry.

MAbs 2014 ;6(6):1464-73

a Laboratoire de spectrométrie de masse des interactions et des systèmes (LSMIS); CNRS - UMR 7140 , Université de Strasbourg , Strasbourg Cedex , France.

Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between 2 samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.
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http://dx.doi.org/10.4161/mabs.36305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623110PMC
September 2015

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

J Cell Sci 2015 Jan 20;128(2):225-31. Epub 2014 Nov 20.

School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, SA, Australia Centre for Molecular Pathology, University of Adelaide, Adelaide, SA 5005, Australia

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.
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http://dx.doi.org/10.1242/jcs.158451DOI Listing
January 2015

The RNA helicases AtMTR4 and HEN2 target specific subsets of nuclear transcripts for degradation by the nuclear exosome in Arabidopsis thaliana.

PLoS Genet 2014 Aug 21;10(8):e1004564. Epub 2014 Aug 21.

Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, UPR 2357, Université de Strasbourg, Strasbourg, France.

The RNA exosome is the major 3'-5' RNA degradation machine of eukaryotic cells and participates in processing, surveillance and turnover of both nuclear and cytoplasmic RNA. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. We show that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the nucleoplasm and appears to be excluded from nucleoli. We have previously shown that the major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products. Here, we demonstrate that HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates such as snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts produced from pseudogenes and intergenic regions. Only a weak accumulation of these exosome substrate targets is observed in mtr4 mutants, suggesting that MTR4 can contribute, but plays rather a minor role for the degradation of non-ribosomal RNAs and cryptic transcripts in Arabidopsis. Consistently, transgene post-transcriptional gene silencing (PTGS) is marginally affected in mtr4 mutants, but increased in hen2 mutants, suggesting that it is mostly the nucleoplasmic exosome that degrades aberrant transgene RNAs to limit their entry in the PTGS pathway. Interestingly, HEN2 is conserved throughout green algae, mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively.
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http://dx.doi.org/10.1371/journal.pgen.1004564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140647PMC
August 2014

The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator of metabolism.

Cell Metab 2010 May 15;11(5):364-78. Epub 2010 Apr 15.

Molecular Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.

Factor inhibiting HIF-1alpha (FIH) is an asparaginyl hydroxylase. Hydroxylation of HIF-alpha proteins by FIH blocks association of HIFs with the transcriptional coactivators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, and improved glucose and lipid homeostasis and are resistant to high-fat-diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, and enhanced insulin sensitivity and are also protected against high-fat-diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.
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http://dx.doi.org/10.1016/j.cmet.2010.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2893150PMC
May 2010