Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX.

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage.

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1(-/-) null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.

Identification of Outer Membrane Proteins Altered in Response to UVC-Radiation in Vibrio parahaemolyticus and Vibrio alginolyticus.

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m(2)) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.

Interleukin-7 compartmentalizes its receptor signaling complex to initiate CD4 T lymphocyte response.

Interleukin (IL)-7 is a central cytokine that controls homeostasis of the CD4 T lymphocyte pool. Here we show on human primary cells that IL-7 binds to preassembled receptors made up of proprietary chain IL-7Ralpha and the common chain gammac shared with IL-2, -4, -9, -15, and -21 receptors. Upon IL-7 binding, both chains are driven in cholesterol- and sphingomyelin-rich rafts where associated signaling proteins Jak1, Jak3, STAT1, -3, and -5 are found to be phosphorylated. Meanwhile the IL-7.IL-7R complex interacts with the cytoskeleton that halts its diffusion as measured by single molecule fluorescence autocorrelated spectroscopy monitored by microimaging. Comparative immunoprecipitations of IL-7Ralpha signaling complex from non-stimulated and IL-7-stimulated cells confirmed recruitment of proteins such as STATs, but many others were also identified by mass spectrometry from two-dimensional gels. Among recruited proteins, two-thirds are involved in cytoskeleton and raft formation. Thus, early events leading to IL-7 signal transduction involve its receptor compartmentalization into membrane nanodomains and cytoskeleton recruitment.

Linking functionally related genes by sensitive and quantitative characterization of genetic interaction profiles.

Describing at a genomic scale how mutations in different genes influence one another is essential to the understanding of how genotype correlates with phenotype and remains a major challenge in biology. Previous studies pointed out the need for accurate measurements of not only synthetic but also buffering interactions in the characterization of genetic networks and functional modules. We developed a sensitive and efficient method that allows such measurements at a genomic scale in yeast. In a pilot experiment (41 genome-wide screens), we quantified the fitness of 140,000 double deletion strains relative to the corresponding single mutants and identified many genetic interactions. In addition to synthetic growth defects (validated experimentally with factors newly identified as genetically interfering with mRNA degradation), most of the identified genetic interactions measured weak epistatic effects. These weak effects, rarely meaningful when considered individually, were crucial to defining specific signatures for many gene deletions and had a major contribution in defining clusters of functionally related genes.

The p21-activated protein kinase inhibitor Skb15 and its budding yeast homologue are 60S ribosome assembly factors.

Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G(1), and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.

In vivo interactome of Helicobacter pylori urease revealed by tandem affinity purification.

In the human gastric bacterium Helicobacter pylori, two metalloenzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease and UreG, one of the accessory proteins for Ni(2+) incorporation into apourease, were taken as baits for tandem affinity purification. The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Furthermore the tandem affinity purification technology was combined with in vivo cross-link to capture transient interactions. The results revealed different populations of urease complexes: (i) urease captured during activation by Ni(2+) ions comprising all the accessory proteins and (ii) urease in association with metabolic proteins involved e.g. in ammonium incorporation and the cytoskeleton. Using UreG as a bait protein, we copurified HypB, the accessory protein for Ni(2+) incorporation into hydrogenase, that is reported to play a role in urease activation. The interactome of HypB partially overlapped with that of urease and revealed interactions with SlyD, which is known to be involved in hydrogenase maturation as well as with proteins implicated in the formation of [Fe-S] clusters present in the small subunit of hydrogenase. In conclusion, this study provides new insight into coupling of ammonium production and assimilation in the gastric pathogen and the intimate link between urease and hydrogenase maturation.

60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes.

During the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes.

Comprehensive proteome analysis of Mycobacterium ulcerans and quantitative comparison of mycolactone biosynthesis.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.

Sequential protein association with nascent 60S ribosomal particles.

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.

Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation.

Previous work suggested that the release of the nucleolar Tif6 from nascent 60 S subunits occurs in the cytoplasm and requires the cytoplasmic EF-2-like GTPase, Efl1. To check whether this release involves an rRNA structural rearrangement mediated by Efl1, we analyzed the rRNA conformation of the GTPase center of 80 S ribosomes in three contexts: wild-type, Deltaefl1 and a dominant suppressor R1 of Deltaefl1. This analysis was restricted to domain II and VI of 25 S rRNA. The rRNA analysis of R1 ribosomes allows us to distinguish the effects due to depletion of Efl1 from the resulting nucleolar deficit of Tif6. Efl1 inhibits the EF-2 GTPase activity, suggesting that the two proteins share a similar ribosome-binding site. The 80 S ribosomes from either type failed to show any difference of conformation in the two rRNA domains analyzed. However, the same analysis performed on the pool of free 60 S subunits reveals several rRNA conformational differences between wild-type and Deltaefl1 subunits, whereas that from the suppressor strain is similar to wild-type. This suggests that the nucleolar deficit of Tif6 during assembly of the 60 S preribosomes is responsible for the changes in rRNA conformation observed in Deltaefl1 60 S subunits. We also purified 60 S preribosomes from the three genetic contexts by TAP-tagging Tif6. The protein content of 60 S preribosomes associated with Tif6p in a Deltaefl1 strain are obtained at a lower yield but have, surprisingly, a protein composition that is a priori similar to that of wild-type and the suppressor strain.

Role of the N-Acetylmuramoyl-l-Alanyl Amidase, AmiA, of Helicobacter pylori in Peptidoglycan Metabolism, Daughter Cell Separation, and Virulence.

The human gastric pathogen, Helicobacter pylori, is becoming increasingly resistant to most available antibiotics. Peptidoglycan (PG) metabolism is essential to eubacteria, hence, an excellent target for the development of new therapeutic strategies. However, our knowledge on PG metabolism in H. pylori remains poor. We have further characterized an isogenic mutant of the amiA gene encoding a N-acetylmuramoyl-l-alanyl amidase. The amiA mutant displayed long chains of unseparated cells, an impaired motility despite the presence of intact flagella and a tolerance to amoxicillin. Interestingly, the amiA mutant was impaired in colonizing the mouse stomach suggesting that AmiA is a valid target in H. pylori for the development of new antibiotics. Using reverse phase high-pressure liquid chromatography, we analyzed the PG muropeptide composition and glycan chain length distribution of strain 26695 and its amiA mutant. The analysis showed that H. pylori lacked muropeptides with a degree of cross-linking higher than dimeric muropeptides. The amiA mutant was also characterized by a decrease of muropeptides carrying 1,6-anhydro-N-acetylmuramic acid residues, which represent the ends of the glycan chains. This correlated with an increase of very long glycan strands in the amiA mutant. It is suggested that these longer glycan strands are trademarks of the division site. Taken together, we show that the low redundancy on genes involved in PG maturation supports H. pylori as an actractive alternative model to study PG metabolism and cell shape regulation.

Plasmin cleaves the juxtamembrane domain and releases truncated species of the urokinase receptor (CD87) from human bronchial epithelial cells.

The three-domain (D1D2D3) urokinase receptor (CD87) is highly susceptible to cleavage within the D1-D2 linker sequence, but also within the juxtamembrane region by yet poorly characterized proteinases, allowing the release of D1 and D2D3 species in various (patho)physiological body fluids. Using immunoblot analysis and ELISA applied to a recombinant soluble CD87 and to CD87-expressing epithelial cells, we establish that exogenous or in situ generated plasmin proteolyzes CD87 in the D1-D2 linker and D3 carboxyterminal sequences, producing a major soluble D2D3 species. Mass spectrometry analysis of the fragmentation of CD87-related synthetic peptides, and aminoterminal sequencing of D2D3 reveal Arg83, Arg89, and Arg281 as residues targeted by plasmin within human CD87.

Differential expression of Ixodes ricinus salivary gland proteins in the presence of the Borrelia burgdorferi sensu lato complex.

In Europe, Ixodes ricinus is the main vector of Lyme borreliosis. Their salivary glands play a critical role in the biological success of ticks. To better understand the cross-talk between Borrelia burgdorferi and tick salivary glands, we analyzed protein expression in the salivary glands of I. ricinus adult ticks that were infected by various strains of the B. burgdorferi sl complex. iTRAQ allowed the identification of more than 120 proteins, providing the first proteomic data pertaining to I. ricinus salivary glands. Among these proteins, only 12 were modulated in the presence of various Borrelia strains. Most of them are up-regulated and are involved in cell defense and protein synthesis and processing. Down-regulated proteins are mostly implicated in the cytoskeleton. The DIGE analysis allowed us to identify 35 proteins and showed the down-regulation of 4 proteins. All 15 proteins were not modulated by all strains. Overall, these observations showed that the presence of Borrelia in tick salivary glands is a factor of stress for the protein machinery, and also that some Borrelia strains produce a dysregulation of cytoskeletal proteins. Interestingly, a protein from Borrelia, OspA, was found in infected salivary glands. The consequence of its presence in salivary glands is discussed. BIOLOGICAL SIGNIFICANCE: Lyme borreliosis is still the most prevalent arthropod-borne disease in the temperate regions of the northern hemisphere. The geographical distribution of Lyme borreliosis is expanding, especially towards higher altitudes and latitudes. Human pathogenic spirochetes causing Lyme borreliosis belong to the B. burgdorferi sensu lato complex. They are extracellular pathogens transmitted to humans through the bite of Ixodes spp. ticks. The bioactive molecules present in tick saliva not only promote tick feeding, but also create an advantageous microenvironment at the tick bite site for survival and replication of Borrelia bacteria. Investigation of the tick-host-pathogen interface would provide new strategies to control tick-borne infections. We chose to analyze the interaction of several strains of the B. burgdorferi sensu lato complex with I. ricinus salivary glands. We also investigated the presence of bacterial proteins in salivary glands. For these purposes, we undertook a proteomic study implying the complementary approaches of iTRAQ and DIGE. Our study allowed identifying several salivary markers of infection that were shown to vary according to the strain. Moreover, OspA, a bacterial protein was shown to be expressed in salivary glands and may be implied in the pathogenicity of some Borrelia strains.

Characterization of the enzymatic activity of Clostridium perfringens TpeL.

TpeL is a toxin produced by Clostridium perfringens which belongs to the large clostridial glucosylating toxin family. It was shown that TpeL modifies Ras using UDP-glucose or UDP-N-acetylglucosamine as cosubstrates (Guttenberg et al., 2012; Nagahama et al., 2011). We confirmed that TpeL preferentially glucosaminates the three isoforms of Ras (cH-Ras, N-Ras, and K-Ras) from UDP-N-acetylglucosamine and to a lower extent Rap1a and R-Ras3, and very weakly Rac1. In contrast to previous report, we observed that Ral was not a substrate of TpeL. In addition, we confirmed by in vitro glucosylation and mass spectrometry that TpeL modifies cH-Ras at Thr35.

Infection by chikungunya virus modulates the expression of several proteins in Aedes aegypti salivary glands.

BACKGROUND: Arthropod-borne viral infections cause several emerging and resurging infectious diseases. Among the diseases caused by arboviruses, chikungunya is responsible for a high level of severe human disease worldwide. The salivary glands of mosquitoes are the last barrier before pathogen transmission. METHODS: We undertook a proteomic approach to characterize the key virus/vector interactions and host protein modifications that occur in the salivary glands that could be responsible for viral transmission by using quantitative two-dimensional electrophoresis. RESULTS: We defined the protein modulations in the salivary glands of Aedes aegypti that were triggered 3 and 5 days after an oral infection (3 and 5 DPI) with chikungunya virus (CHIKV). Gel profile comparisons showed that CHIKV at 3 DPI modulated the level of 13 proteins, and at 5 DPI 20 proteins. The amount of 10 putatively secreted proteins was regulated at both time points. These proteins were implicated in blood-feeding or in immunity, but many have no known function. CHIKV also modulated the quantity of proteins involved in several metabolic pathways and in cell signalling. CONCLUSION: Our study constitutes the first analysis of the protein response of Aedes aegypti salivary glands infected with CHIKV. We found that the differentially regulated proteins in response to viral infection include structural proteins and enzymes for several metabolic pathways. Some may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by arboviruses. For example, proteins involved in blood-feeding such as the short D7, an adenosine deaminase and inosine-uridine preferring nucleoside hydrolase, may favour virus transmission by exerting an increased anti-inflammatory effect. This would allow the vector to bite without the bite being detected. Other proteins, like the anti-freeze protein, may support vector protection.

CCSV-MPase, a novel procoagulant metalloproteinase from Cerastes cerastes venom: purification, biochemical characterization and protein identification.

A procoagulant metalloproteinase called CCSV-MPase was purified from C. cerastes venom by successive chromatographic methods starting with gel-filtration through Sephadex G-75; ion-exchange DEAE-Cellulose A-50; affinity chromatography on Benzamidine Sepharose 6B and RP-HPLC on a C8 column. CCSV-MPase has been isolated to an extent of about tenfolds and its molecular mass was evaluated at 70 kDa by SDS-PAGE. CCSV-MPase hydrolyzes casein and fibrinogene as natural substrates. Its proteolytic activity was inhibited by EDTA and 1.10-phenanthroline, a chelators of bivalent cation metals and Zn(2+) respectively. CCSV-MPase is therefore a Zn(2+)-metalloproteinase with fibrinogenolytic but not hemorrhagic activity. It greatly decreased levels of plasmatic fibrinogen when administered to rats for 24 h. This fibrinogenase hydrolyzes the Bß chain of human fibrinogen in vitro releasing fibrinopeptide B only. LC MS/MS analysis of tryptic fragments of CCSV-MPase demonstrated that it showed some sequence similarities with four other venom metalloproteinases. CCSV-MPase could be considered as a potential therapeutic agent as it is a non-hemorrhagic enzyme and could be useful in thrombotic diseases because of its defibrinogenation of blood.

Identification of outer membrane proteins of Vibrio parahaemolyticus and Vibrio alginolyticus altered in response to γ-irradiation or long-term starvation.

Vibrio parahaemolyticus and Vibrio alginolyticus were subjected to γ-irradiation (0.5 kGy) or starvation by incubation for 8 months in seawater to study modifications in their outer membrane protein patterns. After treatment, outer membrane protein profiles of starved or γ-irradiated bacteria were found to be altered when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Altered proteins were identified by mass spectrometry (MS and MS/MS) and analyses revealed that OmpU can be considered a starvation stress-induced protein. In addition, expression of OtnA, OmpW, OmpA and peptidoglycan-associated lipoprotein decreased to non-detectable levels in starved cells. Furthermore, MltA-interacting protein MipA appeared under γ-irradiation or starvation conditions. Thus, it can be considered to be a γ-irradiation, long-term starvation stress protein in some vibrios.