Multiple pathways for Plasmodium ookinete invasion of the mosquito midgut.

Plasmodium ookinete invasion of the mosquito midgut is a crucial step of the parasite life cycle but little is known about the molecular mechanisms involved. Previously, a phage display peptide library screen identified SM1, a peptide that binds to the mosquito midgut epithelium and inhibits ookinete invasion. SM1 was characterized as a mimotope of an ookinete surface enolase and SM1 presumably competes with enolase, the presumed ligand, for binding to a putative midgut receptor. Here we identify a mosquito midgut receptor that binds both SM1 and ookinete surface enolase, termed "enolase-binding protein" (EBP). Moreover, we determined that Plasmodium berghei parasites are heterogeneous for midgut invasion, as some parasite clones are strongly inhibited by SM1 whereas others are not. The SM1-sensitive parasites required the mosquito EBP receptor for midgut invasion whereas the SM1-resistant parasites invaded the mosquito midgut independently of EBP. These experiments provide evidence that Plasmodium ookinetes can invade the mosquito midgut by alternate pathways. Furthermore, another peptide from the original phage display screen, midgut peptide 2 (MP2), strongly inhibited midgut invasion by P. berghei (SM1-sensitive and SM1-resistant) and Plasmodium falciparum ookinetes, suggesting that MP2 binds to a separate, universal receptor for midgut invasion.

Conformational dissection of Thermomyces lanuginosus lipase in solution.

Lipases are triacyl glycerol acyl hydrolases, which catalyze hydrolysis of esters, esterification and transesterification reactions, among others. Some of these enzymes have a large hydrophobic pocket covered by an alpha-helical mobile surface loop (the lid). Protein-protein interactions can occur through adsorption of two open lids of individual lipases. We investigated the conformation and oligomeric state of Thermomyces lanuginosus lipase (TLL) in solution by spectroscopic and mass spectrometry techniques. Information about oligomerization of this important industrial enzyme is only available for TLL crystals; therefore, we have done a throughout investigation of the conformation of this lipase in solution. SDS-PAGE and mass spectrometry analysis of size-exclusion chromatography eluted fractions indicated the presence of both monomeric and dimeric populations of TLL. The stability of the enzyme upon thermal and guanidine hydrochloride treatment was examined by circular dichroism and fluorescence emission spectroscopy. Small angle x-ray scattering and ion mobility mass spectrometry analysis revealed that TLL is found as a mixture of monomers and dimers at the assayed concentrations. Although previous x-ray diffraction data showed TLL as a dimer in the crystal (PDB: 1DT3), to our knowledge our report is the first evidencing that TLL co-exists as stable dimeric and monomeric forms in solution.

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur.

BACKGROUND: Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. RESULTS: The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3-8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. CONCLUSIONS: Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

Proteomic analysis of two Trypanosoma cruzi zymodeme 3 strains.

Two Trypanosoma cruzi Z3 strains, designated as 3663 and 4167, were previously isolated from insect vectors captured in the Brazilian Amazon region. These strains exhibited different infection patterns in Vero, C6/36, RAW 264.7 and HEp-2 cell lineages, in which 3663 trypomastigote form was much less infective than 4167 ones. A proteomic approach was applied to investigate the differences in the global patterns of protein expression in these two Z3 strains. Two-dimensional (2D) protein maps were generated and certain spots were identified by mass spectrometry (MS). Our analyses revealed a significant difference in the expression profile of different proteins between strains 3663 and 4167. Among them, cruzipain, an important regulator of infectivity. This data was corroborated by flow cytometry analysis using anti-cruzipain antibody. This difference could contribute to the infectivity profiles observed for each strain by in vitro assay using different cell lines.

Malaria parasite invasion of the mosquito salivary gland requires interaction between the Plasmodium TRAP and the Anopheles saglin proteins.

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites.

Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen.

Malaria parasites must undergo development within mosquitoes to be transmitted to a new host. Antivector transmission-blocking vaccines inhibit parasite development by preventing ookinete interaction with mosquito midgut ligands. Therefore, the discovery of novel midgut antigen targets is paramount. Jacalin (a lectin) inhibits ookinete attachment by masking glycan ligands on midgut epithelial surface glycoproteins. However, the identities of these midgut glycoproteins have remained unknown. Here we report on the molecular characterization of an Anopheles gambiae aminopeptidase N (AgAPN1) as the predominant jacalin target on the mosquito midgut luminal surface and provide evidence for its role in ookinete invasion. alpha-AgAPN1 IgG strongly inhibited both Plasmodium berghei and Plasmodium falciparum development in different mosquito species, implying that AgAPN1 has a conserved role in ookinete invasion of the midgut. Molecules targeting single midgut antigens seldom achieve complete abrogation of parasite development. However, the combined blocking activity of alpha-AgAPN1 IgG and an unrelated inhibitory peptide, SM1, against P. berghei was incomplete. We also found that SM1 can block only P. berghei, whereas alpha-AgAPN1 IgG can block both parasite species significantly. Therefore, we hypothesize that ookinetes can evade inhibition by two potent transmission-blocking molecules, presumably through the use of other ligands, and that this process further partitions murine from human parasite midgut invasion models. These results advance our understanding of malaria parasite-mosquito host interactions and guide in the design of transmission-blocking vaccines.

Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile.

The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.

GCN5 acetyltransferase complex controls glucose metabolism through transcriptional repression of PGC-1alpha.

Hormonal and nutrient regulation of hepatic gluconeogenesis mainly occurs through modulation of the transcriptional coactivator PGC-1alpha. The identity of endogenous proteins and their enzymatic activities that regulate the functions and form part of PGC-1alpha complex are unknown. Here, we show that PGC-1alpha is in a multiprotein complex containing the acetyltransferase GCN5. PGC-1alpha is directly acetylated by GCN5 resulting in a transcriptionally inactive protein that relocalizes from promoter regions to nuclear foci. Adenoviral-mediated expression of GCN5 in cultured hepatocytes and in mouse liver largely represses activation of gluconeogenic enzymes and decreases hepatic glucose production. Thus, we have identified the endogenous PGC-1alpha protein complex and provided the molecular mechanism by which PGC-1alpha acetylation by GCN5 turns off the transcriptional and biological function of this metabolic coactivator. GCN5 might be a pharmacological target to regulate the activity of PGC-1alpha, providing a potential treatment for metabolic disorders in which hepatic glucose output is dysregulated.

Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC).

Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes.

Genome annotation of Anopheles gambiae using mass spectrometry-derived data.

BACKGROUND: A large number of animal and plant genomes have been completely sequenced over the last decade and are now publicly available. Although genomes can be rapidly sequenced, identifying protein-coding genes still remains a problematic task. Availability of protein sequence data allows direct confirmation of protein-coding genes. Mass spectrometry has recently emerged as a powerful tool for proteomic studies. Protein identification using mass spectrometry is usually carried out by searching against databases of known proteins or transcripts. This approach generally does not allow identification of proteins that have not yet been predicted or whose transcripts have not been identified. RESULTS: We searched 3,967 mass spectra from 16 LC-MS/MS runs of Anopheles gambiae salivary gland homogenates against the Anopheles gambiae genome database. This allowed us to validate 23 known transcripts and 50 novel transcripts. In addition, a novel gene was identified on the basis of peptides that matched a genomic region where no gene was known and no transcript had been predicted. The amino termini of proteins encoded by two predicted transcripts were confirmed based on N-terminally acetylated peptides sequenced by tandem mass spectrometry. Finally, six sequence polymorphisms could be annotated based on experimentally obtained peptide sequences. CONCLUSION: The peptide sequences from this study were mapped onto the genomic sequence using the distributed annotation system available at Ensembl and can be visualized in the context of all other existing annotations. The strategy described in this paper can be used to correct and confirm genome annotations and permit discovery of novel proteins in a high-throughput manner by mass spectrometry.

A proteomic analysis of salivary glands of female Anopheles gambiae mosquito.

Understanding the development of the malaria parasite within the mosquito vector at the molecular level should provide novel targets for interrupting parasitic life cycle and subsequent transmission. Availability of the complete genomic sequence of the major African malaria vector, Anopheles gambiae, allows discovery of such targets through experimental as well as computational methods. In the female mosquito, the salivary gland tissue plays an important role in the maturation of the infective form of the malaria parasite. Therefore, we carried out a proteomic analysis of salivary glands from female An. gambiae mosquitoes. Salivary gland extracts were digested with trypsin using two complementary approaches and analyzed by LC-MS/MS. This led to identification of 69 unique proteins, 57 of which were novel. We carried out a functional annotation of all proteins identified in this study through a detailed bioinformatics analysis. Even though a number of cDNA and Edman degradation-based approaches to catalog transcripts and proteins from salivary glands of mosquitoes have been published previously, this is the first report describing the application of MS for characterization of the salivary gland proteome. Our approach should prove valuable for characterizing proteomes of parasites and vectors with sequenced genomes as well as those whose genomes are yet to be fully sequenced.

Precursors of novel Gla-containing conotoxins contain a carboxy-terminal recognition site that directs gamma-carboxylation.

Vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamyl residues to gamma-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the gamma-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel gamma-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs gamma-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue "postpeptide" from the Gla-TxXI precursor reduced the K(m) for the reaction of the Conus gamma-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it was positioned at the N- or C-terminal end of the mature toxin. Comparison of the postpeptides to propeptides from other conotoxins suggested some common elements, and amino acid substitutions of these residues perturbed gamma-carboxylation of the Gla-TxXI peptide. The demonstration of a functional and transferable C-terminal postpeptide in these conotoxins indicates the presence of the gamma-carboxylation recognition site within the postpeptide and defines a novel precursor structure for vitamin K-dependent polypeptides. It also provides the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.

A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture.

Posttranslational modifications are major mechanisms of regulating protein activity and function in vertebrate cells. It is essential to obtain qualitative information about posttranslational modification patterns of proteins to understand signal transduction mechanisms in greater detail. However, it is equally important to measure the dynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective. Despite a number of advances, methods to quantitate posttranslational modifications remain difficult to implement due to a number of factors including lack of a generic method, elaborate chemical steps, and requirement for large amounts of sample. We have previously shown that stable isotope-containing amino acids in cell culture (SILAC) can be used to differentially label growing cell populations for quantitation of protein levels. In this report, we extend the use of SILAC as a novel proteomic approach for the relative quantitation of posttranslational modifications such as phosphorylation. We have used SILAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.

Tackling the phosphoproteome: tools and strategies.

Characterization of post-translational modifications in proteins is one of the major tasks that is to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification that regulates enzymatic activity, subcellular localization, complex formation and degradation of proteins. Rapid advances in mass spectrometry instrumentation coupled to the development of analytical methods over the past several years now allow us to investigate the phosphoproteome on a global scale.

Identification of 30 protein species involved in replicative senescence and stress-induced premature senescence.

Exposure of human proliferative cells to subcytotoxic stress triggers stress-induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress-induced premature senescence indicates that, at the level of protein expression, stress-induced premature senescence and replicative senescence are different phenotypes sharing however similarities. In this study, we identified 30 proteins showing changes of expression level specific or common to replicative senescence and/or stress-induced premature senescence. These changes affect different cell functions, including energy metabolism, defense systems, maintenance of the redox potential, cell morphology and transduction pathways.

A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes.

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.

Context-specific effects of fibulin-5 (DANCE/EVEC) on cell proliferation, motility, and invasion. Fibulin-5 is induced by transforming growth factor-beta and affects protein kinase cascades.

Fibulin-5 (FBLN-5; also known as DANCE or EVEC) is an integrin-binding extracellular matrix protein that mediates endothelial cell adhesion; it is also a calcium-dependent elastin-binding protein that scaffolds cells to elastic fibers, thereby preventing elastinopathy in the skin, lung, and vasculature. Transforming growth factor-beta (TGF-beta) regulates the production of cytokines, growth factors, and extracellular matrix proteins by a variety of cell types and tissues. We show here that TGF-beta stimulates murine 3T3-L1 fibroblasts to synthesize FBLN-5 transcript and protein through a Smad3-independent pathway. Overexpression of FBLN-5 in 3T3-L1 cells increased DNA synthesis and enhanced basal and TGF-beta-stimulated activation of ERK1/ERK2 and p38 mitogen-activated protein kinase (MAPK). FBLN-5 overexpression also augmented the tumorigenicity of human HT1080 fibrosarcoma cells by increasing their DNA synthesis, migration toward fibronectin, and invasion through synthetic basement membranes. In stark contrast, FBLN-5 expression was down-regulated in the majority of metastatic human malignancies, particularly in cancers of the kidney, breast, ovary, and colon. Unlike its proliferative response in fibroblasts, FBLN-5 overexpression in mink lung Mv1Lu epithelial cells resulted in an antiproliferative response, reducing their DNA synthesis and cyclin A expression. Moreover, FBLN-5 synergizes with TGF-beta in stimulating AP-1 activity in Mv1Lu cells, an effect that was abrogated by overexpression of dominant-negative versions of either MKK1 or p38 MAPKalpha. Accordingly, both the stimulation and duration of ERK1/ERK2 and p38 MAPK by TGF-beta was enhanced in Mv1Lu cells expressing FBLN-5. Our findings identify FBLN-5 as a novel TGF-beta-inducible target gene that regulates cell growth and motility in a context-specific manner and affects protein kinase activation by TGF-beta. Our findings also indicate that aberrant FBLN-5 expression likely contributes to tumor development in humans.

Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.

Experimental proteome analysis was combined with a genome-wide prediction screen to characterize the protein content of the thylakoid lumen of Arabidopsis chloroplasts. Soluble thylakoid proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry. The identities of 81 proteins were established, and N termini were sequenced to validate localization prediction. Gene annotation of the identified proteins was corrected by experimental data, and an interesting case of alternative splicing was discovered. Expression of a surprising number of paralogs was detected. Expression of five isomerases of different classes suggests strong (un)folding activity in the thylakoid lumen. These isomerases possibly are connected to a network of peripheral and lumenal proteins involved in antioxidative response, including peroxiredoxins, m-type thioredoxins, and a lumenal ascorbate peroxidase. Characteristics of the experimentally identified lumenal proteins and their orthologs were used for a genome-wide prediction of the lumenal proteome. Lumenal proteins with a typical twin-arginine translocation motif were predicted with good accuracy and sensitivity and included additional isomerases and proteases. Thus, prime functions of the lumenal proteome include assistance in the folding and proteolysis of thylakoid proteins as well as protection against oxidative stress. Many of the predicted lumenal proteins must be present at concentrations at least 10,000-fold lower than proteins of the photosynthetic apparatus.