UAP56 associates with DRM2 and is localized to chromatin in Arabidopsis.

Repeated sequence expression and transposable element mobilization are tightly controlled by multilayer processes, which include DNA 5'-cytosine methylation. The RNA-directed DNA methylation (RdDM) pathway, which uses siRNAs to guide sequence-specific directed DNA methylation, emerged specifically in plants. RdDM ensures DNA methylation maintenance on asymmetric CHH sites and specifically initiates de novo methylation in all cytosine sequence contexts through the action of DRM DNA methyltransferases, of which DRM2 is the most prominent. The RdDM pathway has been well described, but how DRM2 is recruited onto DNA targets and associates with other RdDM factors remains unknown. To address these questions, we developed biochemical approaches to allow the identification of factors that may escape genetic screens, such as proteins encoded by multigenic families. Through both conventional and affinity purification of DRM2, we identified DEAD box RNA helicases U2AF56 Associated Protein 56 (UAP56a/b), which are widespread among eukaryotes, as new DRM2 partners. We have shown that, similar to DRM2 and other RdDM actors, UAP56 has chromatin-associated protein properties. We confirmed this association both in vitro and in vivo in reproductive tissues. In addition, our experiments also suggest that UAP56 may exhibit differential distribution in cells depending on plant organ. While originally identified for its role in splicing, our study suggests that UAP56 may also have other roles, and our findings allow us to initiate discussion about its potential role in the RdDM pathway.

Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry.

The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.

A complex small RNA repertoire is generated by a plant/fungal-like machinery and effected by a metazoan-like Argonaute in the single-cell human parasite Toxoplasma gondii.

In RNA silencing, small RNAs produced by the RNase-III Dicer guide Argonaute-like proteins as part of RNA-induced silencing complexes (RISC) to regulate gene expression transcriptionally or post-transcriptionally. Here, we have characterized the RNA silencing machinery and exhaustive small RNAome of Toxoplasma gondii, member of the Apicomplexa, a phylum of animal- and human-infecting parasites that cause extensive health and economic damages to human populations worldwide. Remarkably, the small RNA-generating machinery of Toxoplasma is phylogenetically and functionally related to that of plants and fungi, and accounts for an exceptionally diverse array of small RNAs. This array includes conspicuous populations of repeat-associated small interfering RNA (siRNA), which, as in plants, likely generate and maintain heterochromatin at DNA repeats and satellites. Toxoplasma small RNAs also include many microRNAs with clear metazoan-like features whose accumulation is sometimes extremely high and dynamic, an unexpected finding given that Toxoplasma is a unicellular protist. Both plant-like heterochromatic small RNAs and metazoan-like microRNAs bind to a single Argonaute protein, Tg-AGO. Toxoplasma miRNAs co-sediment with polyribosomes, and thus, are likely to act as translational regulators, consistent with the lack of catalytic residues in Tg-AGO. Mass spectrometric analyses of the Tg-AGO protein complex revealed a common set of virtually all known RISC components so far characterized in human and Drosophila, as well as novel proteins involved in RNA metabolism. In agreement with its loading with heterochromatic small RNAs, Tg-AGO also associates substoichiometrically with components of known chromatin-repressing complexes. Thus, a puzzling patchwork of silencing processor and effector proteins from plant, fungal and metazoan origin accounts for the production and action of an unsuspected variety of small RNAs in the single-cell parasite Toxoplasma and possibly in other apicomplexans. This study establishes Toxoplasma as a unique model system for studying the evolution and molecular mechanisms of RNA silencing among eukaryotes.

The calcium-dependent interaction between S100B and the mitochondrial AAA ATPase ATAD3A and the role of this complex in the cytoplasmic processing of ATAD3A.

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.

The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii.

SUMOylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. Here we show, by characterization of the Toxoplasma gondii SUMO pathway, that the SUMO conjugation system operates in apicomplexan parasites. A gene encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation and release. Various SUMO conjugates were immuno-detected and by means of a global proteomic-based approach, we identified several T. gondii SUMOylated proteins that reveal many diverse cellular processes in which the modification plays a role. More specifically, SUMO conjugates were seen at the tachyzoite surface in response to signaling generated by host cell contact at the time of invasion. Also, under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), we observed the conjugates at the parasitophorous vacuole membrane. The labeling was also at the surface of the mature cysts isolated from parasite-infected mouse brain. Overall, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in T. gondii with initial data indicating that it is likely to play a putative role in host cell invasion and cyst genesis.

The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences.

The ability of living cells to alter their gene expression patterns in response to environmental changes is essential for viability. Oxidative stress represents a common threat for all aerobic life. In normally growing cells, in which hydrogen peroxide generation is transient or pulsed, the antioxidant systems efficiently control its concentration. Intracellular parasites must also protect themselves against the oxidative burst imposed by the host. In this work, we have investigated the role of KMTox, a new histone lysine methyltransferase, in the obligate intracellular parasite Toxoplasma gondii. KMTox is a nuclear protein that holds a High Mobility Group domain, which is thought to recognize bent DNA. The enzyme methylates both histones H4 and H2A in vitro with a great preference for the substrate in reduced conditions. Importantly, KMTox interacts specifically with the typical 2-cys peroxiredoxin-1 and the binding is to some extent enhanced upon oxidation. It appears that the cellular functions that are primarily regulated by the KMTox are antioxidant defences and maintenance of cellular homeostasis. KMTox may regulate gene expression in T. gondii by providing the rapid re-arrangement of chromatin domains and by interacting with the redox-sensor TgPrx1 contribute to establish the antioxidant 'firewall' in T. gondii.

Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway.

Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they further stably associate with PV membranes. In this study, we explored the unusual biochemical behavior of GRA proteins during their trafficking. Conventional chromatography indicated that the GRA proteins form high globular weight complexes within the parasite. To confirm these results, DeltaGRA knocked-out parasites were stably complemented with their respective HA-FLAG tagged GRA2 or GRA5. Purification of the tagged proteins by affinity chromatography showed that within the parasite and the PV soluble fraction, both the soluble GRA2-HA-FLAG and GRA5-HA-FLAG associate with several GRA proteins, the major ones being GRA3, GRA6 and GRA7. Following their insertion into the PV membranes, GRA2-HA-FLAG associated with GRA5 and GRA7 while GRA5-HA-FLAG associated with GRA7 only. Taken together, these data suggest that the GRA proteins form oligomeric complexes that may explain their solubility within the dense granules and the vacuolar matrix by sequestering their hydrophobic domains within the interior of the complex. Insertion into the PV membranes correlates with the decrease of the GRA partners number.

SET8-mediated methylations of histone H4 lysine 20 mark silent heterochromatic domains in apicomplexan genomes.

Posttranslational histone modifications modulate chromatin-templated processes in various biological systems. H4K20 methylation is considered to have an evolutionarily ancient role in DNA repair and genome integrity, while its function in heterochromatin function and gene expression is thought to have arisen later during evolution. Here, we identify and characterize H4K20 methylases of the Set8 family in Plasmodium and Toxoplasma, two medically important members of the protozoan phylum Apicomplexa. Remarkably, parasite Set8-related proteins display H4K20 mono-, di-, and trimethylase activities, in striking contrast to the monomethylase-restricted human Set8. Structurally, few residues forming the substrate-specific channel dictate enzyme methylation multiplicity. These enzymes are cell cycle regulated and focally enriched at pericentric and telomeric heterochromatin in both parasites. Collectively, our findings provide new insights into the evolution of Set8-mediated biochemical pathways, suggesting that the heterochromatic function of the marker is not restricted to metazoans. Thus, these lower eukaryotes have developed a diverse panel of biological stages through their high capacity to differentiate, and epigenetics only begins to emerge as a strong determinant of their biology.

Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii.

Pathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.

Triatoma infestans apyrases belong to the 5'-nucleotidase family.

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bug Triatoma infestans was achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes five N-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5'-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase from Aedes aegypti and with 5'-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5'-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity in T. infestans could represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.

The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression.

Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.

Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of Edman degradation and mass spectrometric techniques.

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.

BRCA1 interacts with acetyl-CoA carboxylase through its tandem of BRCT domains.

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 is a 220-kDa protein that contains a tandem of two BRCA1 C-Terminal (BRCT) domains. Among missense and nonsense BRCA1 mutations responsible for family breast cancer, some are located into the BRCT tandem of BRCA1 coding sequence. In an attempt to understand how BRCT is critical for BRCA1 function, we search for partners of this BRCT tandem of BRCA1. Using a glutathione-S-transferase (GST) pull-down assay with murine cells, we isolated only one major BRCA1-interacting protein, further identified as Acetyl Coenzyme A (CoA) Carboxylase alpha (ACCA). We showed that this interaction is conserved through murine and human species. We also delineated the minimum interacting region as being the whole tandem of BRCT domains. We demonstrated that BRCA1 interacts in vitro and in vivo with ACCA. This interaction is completely abolished by five distinct germline BRCA1 deleterious mutations affecting the BRCT tandem of BRCA1. Interestingly, ACCA originally known as a rate-limiting enzyme for fatty acids biosynthesis, has been recently shown to be over-expressed in breast cancers and considered as a potential target for anti-neoplastic therapy. Furthermore, our observation is making a bridge between the genetic factors involved in susceptibility to breast and ovarian cancers, and environmental factors such as nutrition considered as key elements in the etiology of those cancers.

Syntaxin 7, syntaxin 8, Vti1 and VAMP7 (vesicle-associated membrane protein 7) form an active SNARE complex for early macropinocytic compartment fusion in Dictyostelium discoideum.

The macropinocytic pathway in Dictyostelium discoideum is organized linearly. After actin-driven internalization, fluid material passes sequentially from endosomes to lysosomes, where molecules are degraded and absorbed. Residual material is exocytosed via post-lysosomal compartments. Syntaxin 7 is a SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) protein that is present and active in D. discoideum endosomes [Bogdanovic, Bruckert, Morio and Satre (2000) J. Biol. Chem. 275, 36691-36697]. Here we report the identification of its main SNARE partners by co-immunoprecipitation and MS peptide sequencing. The syntaxin 7 complex contains two co-t-SNAREs [Vti1 (Vps10p tail interactor 1) and syntaxin 8] and a v-SNARE [VAMP7 (vesicle-associated membrane protein 7)] (where t-SNAREs are SNAREs of the target compartment and v-SNAREs are SNAREs present in donor vesicles). In endosomes and in vitro, syntaxin 7, Vti1 and syntaxin 8 form a complex that is able to bind VAMP7. Antibodies to syntaxin 8 and a soluble recombinant VAMP7 fragment both inhibit in vitro reconstituted D. discoideum endosome fusion. The lysosomal content of syntaxin 7, Vti1, syntaxin 8 and VAMP7 is low compared with that in endosomes, implying a highly active recycling or retention mechanism. A likely model is that VAMP7 is a v-SNARE present on vesicles carrying lysosomal enzymes, and that the syntaxin 7-Vti1-syntaxin 8 t-SNARE complex is associated with incoming endocytic material.

Proteomic analysis of human lysosomes: application to monocytic and breast cancer cells.

To date, about fifty lysosomal hydrolases have been identified, and most of them are targeted towards the lysosomes through a specific mannose-6-phosphate (M-6-P) tag. As more lysosomal hydrolases were expected to be discovered, we performed a proteomic study of soluble lysosomal proteins. Human cells were induced to secrete M-6-P proteins which were affinity purified on immobilized M-6-P receptor. The purified proteins were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. Twenty-two proteins were identified, among which 16 were well-known lysosomal hydrolases. The remaining species distributed as follows: epididymis-specific alpha-mannosidase is a new mannosidase homolog, cystatin F and CREG (cellular repressor of E1A-stimulated genes) were previously identified as M-6-P proteins (Journet et al., Electrophoresis 2000, 21, 3411-3419), and the last three, which are not hydrolases, were up to now considered as nonlysosomal. This two-dimensional reference map of human U937 M-6-P proteins was afterwards used for comparison with M-6-P proteins purified either from U937 differentiated into macrophage-like cells, or from human breast cancer MCF7 cells. Phorbol ester induced differentiation of U937 cells led to limited proteolytic cleavage or maturation of a discrete number of hydrolases. Five additional lysosomal hydrolases were identified from MCF7 samples. These results prove the usefulness of such a procedure to analyze the lysosomal content of various cell lines, to discover new M-6-P proteins, as well as to point towards unknown biological processes.