dFMRP and Caprin, translational regulators of synaptic plasticity, control the cell cycle at the Drosophila mid-blastula transition.

The molecular mechanisms driving the conserved metazoan developmental shift referred to as the mid-blastula transition (MBT) remain mysterious. Typically, cleavage divisions give way to longer asynchronous cell cycles with the acquisition of a gap phase. In Drosophila, rapid synchronous nuclear divisions must pause at the MBT to allow the formation of a cellular blastoderm through a special form of cytokinesis termed cellularization. Drosophila Fragile X mental retardation protein (dFMRP; FMR1), a transcript-specific translational regulator, is required for cellularization. The role of FMRP has been most extensively studied in the nervous system because the loss of FMRP activity in neurons causes the misexpression of specific mRNAs required for synaptic plasticity, resulting in mental retardation and autism in humans. Here, we show that in the early embryo dFMRP associates specifically with Caprin, another transcript-specific translational regulator implicated in synaptic plasticity, and with eIF4G, a key regulator of translational initiation. dFMRP and Caprin collaborate to control the cell cycle at the MBT by directly mediating the normal repression of maternal Cyclin B mRNA and the activation of zygotic frühstart mRNA. These findings identify two new targets of dFMRP regulation and implicate conserved translational regulatory mechanisms in processes as diverse as learning, memory and early embryonic development.

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression [corrected].

Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading, migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.

Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens.

Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfenex Expression Technology™ toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfaI gene was identified that produced>99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfaI from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350mg/L at the 1 liter fermentation scale.

Proteomic analysis of zygote and ookinete stages of the avian malaria parasite Plasmodium gallinaceum delineates the homologous proteomes of the lethal human malaria parasite Plasmodium falciparum.

Delineation of the complement of proteins comprising the zygote and ookinete, the early developmental stages of Plasmodium within the mosquito midgut, is fundamental to understand initial molecular parasite-vector interactions. The published proteome of Plasmodium falciparum does not include analysis of the zygote/ookinete stages, nor does that of P. berghei include the zygote stage or secreted proteins. P. gallinaceum zygote, ookinete, and ookinete-secreted/released protein samples were prepared and subjected to Multidimensional protein identification technology (MudPIT). Peptides of P. gallinaceum zygote, ookinete, and ookinete-secreted proteins were identified by MS/MS, mapped to ORFs (> 50 amino acids) in the extent P. gallinaceum whole genome sequence, and then matched to homologous ORFs in P. falciparum. A total of 966 P. falciparum ORFs encoding orthologous proteins were identified; just over 40% of these predicted proteins were found to be hypothetical. A majority of putative proteins with predicted secretory signal peptides or transmembrane domains were hypothetical proteins. This analysis provides a more comprehensive view of the hitherto unknown proteome of the early mosquito midgut stages of P. falciparum. The results underpin more robust study of Plasmodium-mosquito midgut interactions, fundamental to the development of novel strategies of blocking malaria transmission.

Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts.

Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins. Proteins from seven known complexes were identified including MSP-1/7, the low (RAP1/2 and RAP1/3), and high (RhopH1/H2/H3) molecular weight rhoptry complexes, and the invasion motor complex (GAP45/GAP50/myosinA). Remarkably, a large proportion of identified spectra were derived from only 4 proteins: the GPI-anchored proteins MSP-1 and Pf92, the putative GPI-anchored protein Pf113 and RAP-1, the core component of the two RAP complexes. Each of these proteins predominated in high molecular weight species suggesting their aggregation in much larger complexes than anticipated. To demonstrate that the procedure had isolated novel complexes we focussed on MSP-1, which predominated as a distinct species at approximately 500 kDa by BN-PAGE, approximately twice its expected size. Chemical cross-linking supports the existence of a stable MSP-1 oligomer of approximately 500 kDa, probably comprising a highly stable homodimeric species. Our observations also suggests that oligomerization of MSP-1 is likely to occur outside the C-terminal epidermal growth factor (EGF)-like domains. Confirmation of MSP-1 oligomerization, together with the isolation of a number of known complexes by BN-PAGE, makes it highly likely that novel interactions occur amongst members of this proteome.

Strategies for shotgun identification of post-translational modifications by mass spectrometry.

The global identification of post-translationally modified proteins is a difficult challenge that is currently being addressed by many researchers in the field of mass spectrometry (MS)-based proteomics. The ability to identify thousands of proteins by shotgun-based strategies has made the mere idea of a global analysis of a particular protein modification seem reasonable. There has been much progress in the development of methods that make use of shotgun-based protein identification in the analysis of a wide variety of protein modifications, some of which will be discussed here.

Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis.

Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.

Optimizing TiO2-based phosphopeptide enrichment for automated multidimensional liquid chromatography coupled to tandem mass spectrometry.

An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO2. A comparison of TiO2 with other resins used for phosphopeptide enrichment, Fe(III)-IMAC and ZrO2, was also carried out using tryptic digests of both simple and moderately complex protein mixtures; where TiO2 was shown to be superior in performance.

Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway.

Protein phosphorylation has become a focus of many proteomic studies due to the central role that it plays in biology. We combine peptide-based gel-free isoelectric focusing and immobilized metal affinity chromatography to enhance the detection of phosphorylation events within complex protein samples using LC-MS. This method is then used to carry out a quantitative phosphoproteomic analysis of the tumor necrosis factor (TNF) pathway using HeLa cells metabolically labeled with 15N-containing amino acids, where 145 phosphorylation sites were found to be up-regulated upon the activation of the TNF pathway.

Fragile X mental retardation protein controls trailer hitch expression and cleavage furrow formation in Drosophila embryos.

During the cleavage stage of animal embryogenesis, cell numbers increase dramatically without growth, and a shift from maternal to zygotic genetic control occurs called the midblastula transition. Although these processes are fundamental to animal development, the molecular mechanisms controlling them are poorly understood. Here, we demonstrate that Drosophila fragile X mental retardation protein (dFMRP) is required for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the midblastula transition. dFMRP is observed to colocalize with the cytoplasmic RNP body components Maternal expression at 31B (ME31B) and Trailer Hitch (TRAL) in a punctate pattern throughout the cytoplasm of cleavage-stage embryos. Complementary biochemistry demonstrates that dFMRP does not associate with polyribosomes, consistent with their reported exclusion from many cytoplasmic RNP bodies. By using a conditional mutation in small bristles (sbr), which encodes an mRNA nuclear export factor, to disrupt the normal cytoplasmic accumulation of zygotic transcripts at the midblastula transition, we observe the formation of giant dFMRP/TRAL-associated structures, suggesting that dFMRP and TRAL dynamically regulate RNA metabolism at the midblastula transition. Furthermore, we show that dFMRP associates with endogenous tral mRNA and is required for normal TRAL protein expression and localization, revealing it as a previously undescribed target of dFMRP control. We also show genetically that tral itself is required for cleavage furrow formation. Together, these data suggest that in cleavage-stage Drosophila embryos, dFMRP affects protein expression by controlling the availability and/or competency of specific transcripts to be translated.

Distinct protein classes including novel merozoite surface antigens in Raft-like membranes of Plasmodium falciparum.

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.

Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA.

The interaction of activators with mediator has been proposed to stimulate the assembly of RNA polymerase II (Pol II) preinitiation complexes, but there have been few tests of this model. The finding that the major adenovirus E1A and mitogen-activated protein kinase-phosphorylated Elk1 activation domains bind to Sur2 uniquely among the metazoan mediator subunits and the development of transcriptionally active nuclear extracts from WT and sur2-/- embryonic stem cells, reported here, allowed a direct test of the model. We found that whereas VP16, E1A, and phosphorylated Elk1 activation domains each stimulate binding of mediator, Pol II, and general transcription factors to promoter DNA in extracts from WT cells, only VP16 stimulated their binding in extracts from sur2-/- cells. This stimulation of mediator, Pol II, and general transcription factor binding to promoter DNA correlated with transcriptional activation by these activators in WT and mutant extracts. Because the mutant mediator was active in reactions with the VP16 activation domain, the lack of activity in response to the E1A and Elk1 activation domains was not due to loss of a generalized mediator function, but rather the inability of the mutant mediator to be bound by E1A and Elk1. These results directly demonstrate that the interaction of activation domains with mediator stimulates preinitiation complex assembly on promoter DNA.

Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast.

The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs. The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms. Here we show that pheromone signaling induces Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway. Degradation requires Fus3 kinase activity and a MAPK phosphorylation site in Tec1 at threonine 273. Fus3 associates with Tec1 in unstimulated cells, and active Fus3 phosphorylates Tec1 on T273 in vitro. Destruction of Tec1 requires the F box protein Dia2 (Digs-into-agar-2), and Cdc53, the Cullin of SCF (Skp1-Cdc53-F box) ubiquitin ligases. Notably, mutation of the phosphoacceptor site in Tec1, deletion of FUS3, or deletion of DIA2 results in a loss of signaling specificity such that pheromone pathway signaling erroneously activates filamentation pathway gene expression and invasive growth. Signal-induced destruction of a transcription factor for a competing pathway provides a mechanism for signaling specificity.

Transcription control by E1A and MAP kinase pathway via Sur2 mediator subunit.

Sur2 is a metazoan Mediator subunit that interacts with the adenovirus E1A protein and functions in a mitogen-activated protein kinase pathway required for vulva development in Caenorhabditis elegans. We generated sur2-/- embryonic stem cells to analyze its function as a mammalian Mediator component. Our results show that Sur2 forms a subcomplex of the Mediator with two other subunits, TRAP/Med100 and 95. Knock-out of Sur2 prevents activation by E1A-CR3 and the mitogen-activated protein kinase-regulated ETS transcription factor Elk-1, but not by multiple other transcription factors. These results imply that specific activation domains stimulate transcription by binding to distinct Mediator subunits. Activation by E1A and Elk-1 requires recruitment of Mediator to a promoter by binding to its Sur2 subunit.