Secretome Proteins Research Articles

Pathway-Based Biomarker Search by High-Throughput Proteomics Profiling of Secretomes

An efficient means for the identification of prognostic and predictive biomarkers is essential in today's cancer management. A new approach toward biomarker discovery has therefore been proposed, where pathways instead of individual proteins would be monitored and targeted. Recently, the 'secretome', a biological fluid that may be enriched with secreted and/or shed proteins from adjacent disease-relevant cancer cells, has been targeted for biomarker discovery. We describe a novel method for secretome analysis using "stacking gels", label-free relative quantitation, and pathway analysis. The protocol presented here increases the throughput of secretome analysis by approximately 1 order of magnitude compared to earlier methodologies. In the first application, six cancer cell lines from three different tissues were studied. The global secretome data sets obtained were analyzed using pathway analysis software to attempt integrating the experimental findings into a cellular signaling context. This suggested that several secretome proteins might be interconnected with intracellular canonical pathways. This, in turn, may eventually allow the use of secretomes for discovery of pathway-based biomarkers. When this strategy was applied to two breast cancer cell lines, it appeared that the IGF signaling and the plasminogen activating system may be differentially regulated in invasive breast cancer, but this remains speculative until it is verified in a clinical setting. In summary, the methodology proposed optimizes cell culture with sample fractionation and LC-MS to obtain the highest yield from cultured cell secretomes, with a focus on rational biomarker discovery through putative linkage with cancer relevant pathways.

Mass spectrometric and computational analysis of cytokine‐induced alterations in the astrocyte secretome

The roles of astrocytes in the CNS have been expanding beyond the long held view of providing passive, supportive functions. Recent evidence has identified roles in neuronal development, extracellular matrix maintenance, and response to inflammatory challenges. Therefore, insights into astrocyte secretion are critically important for understanding physiological responses and pathological mechanisms in CNS diseases. Primary astrocyte cultures were treated with inflammatory cytokines for either a short (1 day) or sustained (7 days) exposure. Increased interleukin-6 secretion, nitric oxide production, cyclooxygenase-2 activation, and nerve growth factor (NGF) secretion confirmed the astrocytic response to cytokine treatment. MS/MS analysis, computational prediction algorithms, and functional classification were used to compare the astrocyte protein secretome from control and cytokine-exposed cultures. In total, 169 secreted proteins were identified, including both classically and nonconventionally secreted proteins that comprised components of the extracellular matrix and enzymes involved in processing of glycoproteins and glycosaminoglycans. Twelve proteins were detected exclusively in the secretome from cytokine-treated astrocytes, including matrix metalloproteinase-3 (MMP-3) and members of the chemokine ligand family. This compilation of secreted proteins provides a framework for identifying factors that influence the biochemical environment of the nervous system, regulate development, construct extracellular matrices, and coordinate the nervous system response to inflammation.

Proteomic analysis of alterations in the secretome of Arabidopsis thaliana suspension cells subjected to nutritional phosphate deficiency

A proteomic approach was applied to compare the secretome (culture filtrate proteome) of phosphate-sufficient (+Pi) and Pi-deficient (-Pi) Arabidopsis thaliana suspension cell cultures. Secretomes harvested from the +Pi and -Pi cells yielded dissimilar 2-DE maps. PMF via MALDI-TOF MS resulted in the identification of 50 protein spots representing 37 discrete proteins having unique gene identities. A total of 24 Pi-starvation responsive proteins were identified, with 18 of these being up-regulated and six down-regulated. Secreted proteins up-regulated by the -Pi cells included a ribonuclease involved in Pi scavenging from extracellular nucleic acids, as well as enzymes of cell wall modification, proteolysis, pathogen responses, and ROS metabolism. Enzyme activity assays and immunoblotting demonstrated that a pair of purple acid phosphatase isoforms having subunit M(r)s of 65 and 55 kDa was also secreted by the -Pi cells. Semiquantitative RT-PCR was used to assess the relationship between mRNA levels and relative amounts of selected secretome proteins. The results indicate that transcriptional control is but one of many factors contributing to Arabidopsis Pi starvation responses, and highlight the importance of parallel biochemical/proteomic studies of -Pi plants.

Distinctive attributes for predicted secondary structures at terminal sequences of non-classically secreted proteins from proteobacteria

Abstract C- and N-terminal sequences (64 amino acid residues each) of 89 non-classically secreted type I, type III and type IV proteins (Swiss-Prot/TrEMBL) from proteobacteria were transformed into predicted secondary structures. Multivariate analysis of variance (MANOVA) confirmed the significance of location (C- or N-termini) and secretion type as essential factors in respect of quantitative representations of structured (a-helices, b-strands) and unstructured (coils) elements. The profiles of secondary structures were transcripted using unequal property values for helices, strands and coils and corresponding numerical vectors (independent variables) were subjected to multiple discriminant analysis with the types of secreted proteins as the dependent variables. The set of strong predictor variables (21 property values located at the region of 2–49 residues from the C-termini) was capable to classify all three types of non-classically secreted proteins with an accuracy of 93.3% for originally and 89.9% for cross-validated (leave-one-out procedure) grouped cases. The average error rate (0.137 ± 0.015) of k-fold (k = 3; 4; 6; 8; 10; 89) cross validation affirmed an acceptable prediction accuracy of defined discriminant functions with regard to the types of non-classically secreted proteins. The proposed prediction tool could be used to specify the secretome proteins from genomic sequences as well as to assess the compatibility between secretion pathways and secretion substrates of proteobacteria.

A Plasmodium falciparum Host-Targeting Motif Functions in Export during Blood Stage Infection of the Rodent Malarial Parasite Plasmodium berghei

Plasmodium falciparum (P. falciparum) secretes hundreds of proteins—including major virulence proteins—into the host erythrocyte. In order to reach the host cytoplasm, most P. falciparum proteins contain an N terminal host-targeting (HT) motif composed of 11 amino acids. In silico analyses have suggested that the HT motif is conserved throughout the Plasmodium species but experimental evidence only exists for P. falciparum. Here, we show that in the rodent malaria parasite Plasmodium berghei (P. berghei) a reporter-like green fluorescent protein expressed by the parasite can be exported to the erythrocyte cytoplasm in a HT-specific manner. This provides the first experimental proof that the HT motif can function as a signal for protein delivery to the erythrocyte across Plasmodium species. Further, it suggests that P. berghei may serve as a model for validation of P. falciparum secretome proteins. We also show that tubovesicular membranes extend from the vacuolar parasite into the erythrocyte cytoplasm and speculate that these structures may facilitate protein export to the erythrocyte.

The Zebrafish Secretome

The secretome is a functionally rich proteome subset, including cellular membrane and extracellular proteins processed through the secretory pathway. In this study, Danio rerio and Homo sapiens RefSeq proteins were analyzed with SignalP, TargetP, Phobius, and pTarget algorithms. About 16.5% of the zebrafish proteome and 17.0% of the human proteome possessed predicted N-terminal signal sequences. Nearly half of these proteins were subsequently classified as soluble, as they lacked predicted transmembrane domains. The soluble proteins were further subclassified, predicting 1345 (3.8%) zebrafish and 1207 (3.2%) human proteins as extracellular. Comparison of the zebrafish and human soluble secretome proteins identified 372 as orthologs, on the basis of reciprocal BLAST best hits. The computational characterization of the zebrafish proteins found many more members of the secretome than annotated in the SwissProt database. Only 180 of the 2078 zebrafish SwissProt protein entries, and 995 of the 19,294 human SwissProt protein entries were annotated with secreted protein locales. A specific investigation of the fibroblast growth factor and matrix metalloproteinase (MMP) protein families confirmed the prediction data and generated annotation of three additional putative MMP zebrafish proteins. This study presents the first known published description of the zebrafish secretome since the completion of the zebrafish genome sequencing project.

Cardioinductive Network Guiding Stem Cell Differentiation Revealed by Proteomic Cartography of Tumor Necrosis Factor α-Primed Endodermal Secretome

In the developing embryo, instructive guidance from the ventral endoderm secures cardiac program induction within the anterolateral mesoderm. Endoderm-guided cardiogenesis, however, has yet to be resolved at the proteome level. Here, through cardiopoietic priming of the endoderm with the reprogramming cytokine tumor necrosis factor alpha (TNFalpha), candidate effectors of embryonic stem cell cardiac differentiation were delineated by comparative proteomics. Differential two-dimensional gel electrophoretic mapping revealed that more than 75% of protein species increased >1.5-fold in the TNFalpha-primed versus unprimed endodermal secretome. Protein spot identification by linear ion trap quadrupole (LTQ) tandem mass spectrometry (MS/MS) and validation by shotgun LTQ-Fourier transform MS/MS following multidimensional chromatography mapped 99 unique proteins from 153 spot assignments. A definitive set of 48 secretome proteins was deduced by iterative bioinformatic screening using algorithms for detection of canonical and noncanonical indices of secretion. Protein-protein interaction analysis, in conjunction with respective expression level changes, revealed a nonstochastic TNFalpha-centric secretome network with a scale-free hierarchical architecture. Cardiovascular development was the primary developmental function of the resolved TNFalpha-anchored network. Functional cooperativity of the derived cardioinductive network was validated through direct application of the TNFalpha-primed secretome on embryonic stem cells, potentiating cardiac commitment and sarcomerogenesis. Conversely, inhibition of primary network hubs negated the procardiogenic effects of TNFalpha priming. Thus, proteomic cartography establishes a systems biology framework for the endodermal secretome network guiding stem cell cardiopoiesis.

Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts

BackgroundFrankia sp. strains, the nitrogen-fixing facultative endosymbionts of actinorhizal plants, have long been proposed to secrete hydrolytic enzymes such as cellulases, pectinases, and proteases that may contribute to plant root penetration and formation of symbiotic root nodules. These or other secreted proteins might logically be involved in the as yet unknown molecular interactions between Frankia and their host plants. We compared the genome-based secretomes of three Frankia strains representing diverse host specificities. Signal peptide detection algorithms were used to predict the individual secretomes of each strain, and the set of secreted proteins shared among the strains, termed the core Frankia secretome. Proteins in the core secretome may be involved in the actinorhizal symbiosis.ResultsThe Frankia genomes have conserved Sec (general secretory) and Tat (twin arginine translocase) secretion systems. The potential secretome of each Frankia strain comprised 4–5% of the total proteome, a lower percentage than that found in the genomes of other actinobacteria, legume endosymbionts, and plant pathogens. Hydrolytic enzymes made up only a small fraction of the total number of predicted secreted proteins in each strain. Surprisingly, polysaccharide-degrading enzymes were few in number, especially in strain CcI3, with more esterolytic, lipolytic and proteolytic enzymes having signal peptides. A total of 161 orthologous proteins belong to the core Frankia secretome. Of these, 52 also lack homologs in closely related actinobacteria, and are termed "Frankia-specific." The genes encoding these conserved secreted proteins are often clustered near secretion machinery genes.ConclusionThe predicted secretomes of Frankia sp. are relatively small and include few hydrolases, which could reflect adaptation to a symbiotic lifestyle. There are no well-conserved secreted polysaccharide-degrading enzymes present in all three Frankia genomes, suggesting that plant cell wall polysaccharide degradation may not be crucial to root infection, or that this degradation varies among strains. We hypothesize that the relative lack of secreted polysaccharide-degrading enzymes in Frankia reflects a strategy used by these bacteria to avoid eliciting host defense responses. The esterases, lipases, and proteases found in the core Frankia secretome might facilitate hyphal penetration through the cell wall, release carbon sources, or modify chemical signals. The core secretome also includes extracellular solute-binding proteins and Frankia-specific hypothetical proteins that may enable the actinorhizal symbiosis.

The Mammary Epithelial Cell Secretome and Its Regulation by Signal Transduction Pathways

Extracellular proteins released by mammary epithelial cells are critical mediators of cell communication, proliferation, and organization, yet the actual spectrum of proteins released by any given cell (the secretome) is poorly characterized. To define the set of proteins secreted by human mammary epithelial cells (HMEC), we combined analytical and computational approaches to define a secretome protein set based upon probable biological significance. Analysis of HMEC-conditioned medium by liquid chromatography-mass spectrometry resulted in identification of 889 unique proteins, of which 151 were found to be specifically enriched in the extracellular compartment when compared with a database of proteins expressed in whole HMEC lysates. Additional high mass accuracy analysis revealed 36 proteins whose extracellular abundance increased after treatment with phorbol ester (PMA), a protein kinase C agonist and general secretagogue. Many of the PMA stimulated proteins have been reported to be aberrantly expressed in human cancers and appear to be coregulated as multigene clusters. By inhibiting PMA-mediated transactivation of the epidermal growth factor receptor (EGFR), a pathway critically required for normal HMEC function, we found that the secretion of specific matrix metalloproteases was also coordinately regulated through EGFR transactivation. This study demonstrates a tiered strategy by which extracellular proteins can be identified and progressively assigned to classes of increasing confidence and regulatory importance.

Embryology in the era of proteomics

Currently, relatively little is known regarding the protein production of mammalian embryos. Unlike the genome, the proteome itself is dynamic reflecting both internal and external environmental stimuli. Until now the lack of sensitivity has remained a stumbling block for the global introduction of proteomics into the field of mammalian embryology. However, new developments in mass spectrometry have been revolutionary, utilizing protein profiling and peptide sequencing to elucidate underlying biological processes. The sensitivity of these platforms have allowed for the development of new protocols that are capable of profiling the proteome of individual mammalian oocytes and embryos. This information is fundamental to unravelling the complexity of embryo physiology including the dialogue between the developing embryo and its maternal environment. Such proteomic approaches are also assisting in the optimization of ART techniques, including oocyte cryopreservation and in vitro maturation. Embryo selection for transfer is another area of ART that should benefit in this era of proteomics. Currently, mammalian embryos are selected for transfer based on morphological grading systems. Although of great value, analysis of morphology alone cannot determine the embryo's physiological state or chromosomal complement. Subsequently, there is a need to identify in culture those embryos with the highest implantation potential. Proteomic analysis of the embryonic secretome (proteins produced by the embryo and secreted into the surrounding medium) followed by the identification of specific proteins critical for implantation, may lead to the development of a non-invasive viability assay to assist in the selection of embryos for transfer.

Direct selection and phage display of a Gram-positive secretome

A phage display system for direct selection, identification, expression and purification of bacterial secretome proteins has been developed.

Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

BackgroundThe secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2-) during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax.ResultsA total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites), lipoprotein signal peptides (13 have SpII sites), and N-terminal membrane helices (9 have transmembrane helices). The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices.Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa) of protective antigen (PA) were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase.ConclusionThis is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and accumulation may be relevant in elucidation of the progression of pathogenicity, identification of therapeutics and diagnostic markers, and vaccine development. This study also adds to the continuously growing list of identified Bacillus anthracis secretome proteins.

Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities.

The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulence plasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomic approach. More than 400 protein spots, representing the products of 64 genes, were identified, and a unique pattern of protein relative abundance with respect to the presence of the virulence plasmids was revealed. In minimal medium under high CO(2) tension, conditions considered to simulate those encountered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carried genes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5 pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2 plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic medium not supplemented with CO(2), the virulent and avirulent B. anthracis strains manifest very similar protein signatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of gene products attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome. Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulence determinants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification factors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO(2)-responsive chromosome- and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.

Difference in Mass Analysis Using Labeled Lysines (DIMAL-K): A New, Efficient Proteomic Quantification Method Applied to the Analysis of Astrocytic Secretomes

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

Comparative Secretome Analyses of Three Bacillus anthracis Strains with Variant Plasmid Contents

Bacillus anthracis, the causative agent of anthrax, secretes numerous proteins into the extracellular environment during infection. A comparative proteomic approach was employed to elucidate the differences among the extracellular proteomes (secretomes) of three isogenic strains of B. anthracis that differed solely in their plasmid contents. The strains utilized were the wild-type virulent B. anthracis RA3 (pXO1(+) pXO2(+)) and its two nonpathogenic derivative strains: the toxigenic, nonencapsulated RA3R (pXO1(+) pXO2(-)) and the totally cured, nontoxigenic, nonencapsulated RA3:00 (pXO1(-) pXO2(-)). Comparative proteomics using two-dimensional gel electrophoresis followed by computer-assisted gel image analysis was performed to reveal unique, up-regulated, or down-regulated secretome proteins among the strains. In total, 57 protein spots, representing 26 different proteins encoded on the chromosome or pXO1, were identified by peptide mass fingerprinting. S-layer-derived proteins, such as Sap and EA1, were most frequently observed. Many sporulation-associated enzymes were found to be overexpressed in strains containing pXO1(+). This study also provides evidence that pXO2 is necessary for the maximal expression of the pXO1-encoded toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). Several newly identified putative virulence factors were observed; these include enolase, a high-affinity zinc uptake transporter, the peroxide stress-related alkyl hydroperoxide reductase, isocitrate lyase, and the cell surface protein A.