Céline Henry

The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry.

Double‐stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 Å X‐ray structure of a picobirnavirus—an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two‐fold symmetric dimers of a coat protein (CP) with a new 3D‐fold. We show that, as many non‐enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post‐translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120‐subunits capsid has evolved animal cell invasion properties.

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition.

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post‐translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.

Analysis of the serine/threonine/tyrosine phosphoproteome of the pathogenic bacterium Listeria monocytogenes reveals phosphorylated proteins related to virulence.

Phosphorylation is the most common and widely studied post‐translational protein modification in bacteria. It plays an important role in all kinds of cellular processes and controls key regulatory mechanisms, including virulence in certain pathogens. To gain insight into the role of protein phosphorylation in the pathogen Listeria monocytogenes, the serine (Ser), threonine (Thr) and tyrosine (Tyr) phosphoproteome of this bacterium was determined. We used the “gel free” proteomic approach with high accuracy mass spectrometry after enrichment of phosphopeptides. A total of 143 sites of phosphorylation were clearly identified, on 155 unique peptides of 112 phosphoproteins. The Ser/Thr/Tyr phosphorylation site distribution was 93:43:7. All identified phosphopeptides are monophosphorylated, except one and many identified phosphoproteins are related to virulence, translation, phosphoenolpyruvate:sugar phosphotransferase system, glycolysis and stress response. A description of these phosphoproteins is provided together with a comparison of the phosphosites in the L. monocytogenes proteins and in their homologues of other bacteria for which the phosphoproteome has been determined. Compared with the previous studies, we noticed a more extended conservation of the phosphorylation sites in glycolytic enzymes as well as ribosomal proteins.

The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner

ABSTRACT The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely α-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and ∼7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative α-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.

Blotched Snakehead Virus Is a New Aquatic Birnavirus That Is Slightly More Related to Avibirnavirus Than to Aquabirnavirus

ABSTRACT By different approaches, we characterized the birnavirus blotched snakehead virus (BSNV). The sequence of genomic segment A revealed the presence of two open reading frames (ORFs): a large ORF with a 3,207-bp-long nucleotide sequence and a 417-nucleotide-long small ORF located within the N-terminal half of the large ORF, but in a different reading frame. The large ORF was found to encode a polyprotein cotranslationally processed by the viral protease VP4 to generate pVP2 (the VP2 precursor), a 71-amino-acid-long peptide ([X]), VP4, and VP3. The two cleavage sites at the [X]-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing. We showed that the processing of pVP2 generated VP2 and several small peptides (amino acids [aa] 418 to 460, 461 to 467, 468 to 474, and 475 to 486). Two of these peptides (aa 418 to 460 and 475 to 486) were positively identified in the viral particles with 10 additional peptides derived from further processing of the peptide aa 418 to 460. The results suggest that VP4 cleaves multiple Pro-X-Ala↓Ala motifs, with the notable exception of the VP4-VP3 junction. Replacement of the members of the predicted VP4 catalytic dyad (Ser-692 and Lys-729) confirmed their indispensability in the polyprotein processing. The genomic segment B sequence revealed a single large ORF encoding a putative polymerase, VP1. Our results demonstrate that BSNV should be considered a new aquatic birnavirus species, slightly more related to IBDV than to IPNV.

Glycogen synthase kinase 3‐mediated voltage‐dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation

Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early mitochondrial impairment and late hepatocyte cell death. However, mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high‐fat diet, and in vitro models of lipotoxicity, we show that outer mitochondrial membrane permeability is altered and identified a posttranslational modification of voltage‐dependent anion channel (VDAC), a membrane channel and NADH oxidase, as a cause of early mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. Conclusion: VDAC acts as an early sensor of lipid toxicity and its GSK3‐mediated phosphorylation status controls outer mitochondrial membrane permeabilization in hepatosteatosis. (HEPATOLOGY 2013)

Structural Peptides of a Nonenveloped Virus Are Involved in Assembly and Membrane Translocation

ABSTRACT The capsid of infectious bursal disease virus (IBDV), a nonenveloped virus of the family Birnaviridae, has a T=13l icosahedral shell constituted by a single protein, VP2, and several disordered peptides, all derived from the precursor pVP2. In this study, we show that two of the peptides, pep11 and pep46, control virus assembly and cell entry. Deletion of pep11 or even simple substitution of most of its residues blocks the capsid morphogenesis. Removal of pep46 also prevents capsid assembly but leads to the formation of subviral particles formed by unprocessed VP2 species. Fitting with the VP2 atomic model into three-dimensional reconstructions of these particles demonstrates that the presence of uncleaved pep46 causes a steric hindrance at the vertices, blocking fivefold axis formation. Mutagenesis of the pVP2 maturation sites confirms that C terminus processing is necessary for VP2 to acquire the correct icosahedral architecture. All peptides present on virions are accessible to proteases or biochemical labeling. One of them, pep46, is shown to induce large structural rearrangements in liposomes and to destabilize target membranes, demonstrating its implication in cell entry.

Goat αs1-casein genotype affects milk fat globule physicochemical properties and the composition of the milk fat globule membrane

Milk fat secretion is a complex process that initiates in the endoplasmic reticulum of the mammary epithelial cell by the budding of lipid droplets. Lipid droplets are finally released as fat globules in milk enveloped by the apical plasma membrane of the mammary epithelial cell. The milk fat globule membrane (MFGM) thus comprises membrane-specific proteins and polar lipids (glycerophospholipids and sphingolipids) surrounding a core of neutral lipids (mainly triacylglycerols and cholesterol esters). We have recently described major proteins of the MFGM in the goat and we have highlighted prominent differences between goats and bovine species, especially regarding lactadherin, a major MFGM protein. Here, we show that, in the goat species, the well-documented genetic polymorphism at the α(s1)-casein (CSN1S1) locus affects both structure and composition of milk fat globules. We first evidenced that both milk fat globule size and ζ-potential are related to the α(s1)-casein genotype. At midlactation, goats displaying strong genotypes for α(s1)-casein (A/A goats) produce larger fat globules than goats with a null genotype at the CSN1S1 locus (O/O goats). A linear relationship (R(2)=0.75) between fat content (g/kg) in the milk and diameter of fat globules (μm) was established. Moreover, we found significant differences with regard to MFGM composition (including both polar lipids and MFGM proteins) from goats with extreme genotype at the CSN1S1 locus. At midlactation, the amount of polar lipids is significantly higher in the MFGM from goats with null genotypes for α(s1)-casein (O/O goats; 5.97±0.11mg/g of fat; mean ± standard deviation) than in the MFGM from goats with strong genotypes for α(s1)-casein (A/A goats; 3.96±0.12mg/g of fat; mean ± standard deviation). Two MFGM-associated proteins, namely lactadherin and stomatin, are also significantly upregulated in the MFGM from goats with null genotype for α(s1)-casein at early lactation. Our findings are discussed with regard to techno-functional properties and nutritional value of goat milk. In addition, the genetic polymorphism in the goat species appears to be a tool to provide clues to the lipid secretion pathways in the mammary epithelial cell.

Bacillus subtilis serine/threonine protein kinase YabT is involved in spore development via phosphorylation of a bacterial recombinase.

We characterized YabT, a serine/threonine kinase of the Hanks family, from Bacillus subtilis. YabT is a putative transmembrane kinase that lacks the canonical extracellular signal receptor domain. We demonstrate that YabT possesses a DNA‐binding motif essential for its activation. In vivo YabT is expressed during sporulation and localizes to the asymmetric septum. Cells devoid of YabT sporulate more slowly and exhibit reduced resistance to DNA damage during sporulation. We established that YabT phosphorylates DNA‐recombinase RecA at the residue serine 2. A non‐phosphorylatable mutant of RecA exhibits the same phenotype as the ΔyabT mutant, and a phosphomimetic mutant of RecA complements ΔyabT, suggesting that YabT acts via RecA phosphorylation in vivo. During spore development, phosphorylation facilitates the formation of transient and mobile RecA foci that exhibit a scanning‐like movement associated to the nucleoid in the mother cell. In some cells these foci persist at the end of spore development. We show that persistent RecA foci, which presumably coincide with irreparable lesions, are mutually exclusive with the completion of spore morphogenesis. Our results highlight similarities between the bacterial serine/threonine kinase YabT and eukaryal kinases C‐Abl and Mec1, which are also activated by DNA, and phosphorylate proteins involved in DNA damage repair.

Characterisation of the potential SNARE proteins relevant to milk product release by mouse mammary epithelial cells

Casein micelles and fat globules are essential components of milk and are both secreted at the apical side of mammary epithelial cells during lactation. Milk fat globules are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. SNARE proteins are known to take part in cellular membrane trafficking and in exocytosis events in many cell types and we therefore attempted to identify those relevant to casein secretion. With this aim, we performed a detailed analysis of their expression by RT-PCR in both whole mouse mammary gland and in purified mammary acini at various physiological stages, as well as in the HC11 cell line. The expression of some regulatory proteins involved in SNARE complex formation such as Munc-13, Munc-18 and complexins was also explored. The amount of certain SNAREs appeared to be regulated depending on the physiological stage of the mammary gland. Co-immunoprecipitation experiments indicated that SNAP-23 interacted with syntaxin-6, -7 and -12, as well as with VAMP-3, -4 and -8 in mammary epithelial cells during lactation. Finally, the subcellular localisation of candidate SNAREs in these cells was determined both by indirect immunofluorescence and immunogold labelling. The present work provides important new data concerning SNARE proteins in mammary epithelial cells and points to SNAP-23 as a potential central player for the coupling of casein and milk fat globule secretion during lactation.