Comparative Secretome Analysis Research Articles

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

BackgroundOur dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world's second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.ResultsAnalyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.ConclusionOur work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.

Tumour-derived SPARC drives vascular permeability and extravasation through endothelial VCAM1 signalling to promote metastasis.

Disruption of the endothelial barrier by tumour-derived secreted factors is a critical step in cancer cell extravasation and metastasis. Here, by comparative proteomic analysis of melanoma secretomes, we identify the matricellular protein SPARC as a novel tumour-derived vascular permeability factor. SPARC deficiency abrogates tumour-initiated permeability of lung capillaries and prevents extravasation, whereas SPARC overexpression enhances vascular leakiness, extravasation and lung metastasis. SPARC-induced paracellular permeability is dependent on the endothelial VCAM1 receptor and p38 MAPK signalling. Blocking VCAM1 impedes melanoma-induced endothelial permeability and extravasation. The clinical relevance of our findings is highlighted by high levels of SPARC detected in tumour from human pulmonary melanoma lesions. Our study establishes tumour-produced SPARC and VCAM1 as regulators of cancer extravasation, revealing a novel targetable interaction for prevention of metastasis.

Comparative secretome analysis of Fusarium sp. Q7-31T during liquid fermentation using oat straw as a carbon source

Q7-31T was screened from seven strains with high plant cell wall (PCW)-degrading enzyme activities and identified as Fusarium sp. based on the morphological characteristics and internal transcribed spacer (ITS) rDNA sequence analysis. The protein composition of the secretome produced by Q7-31T grown in a liquid medium with oat straw powders or glucoses as a carbon source was investigated using two-dimensional electrophoresis coupled with tandem mass spectrometry. A total of 28 protein species were identified among 115 spots that only existed or had a larger quantity (threefold or more) in the induction medium. As expected, most of them were potentially involved in PCW degradation. Six different superfamilies of glucoside hydrolases (GH5, GH7, GH10, GH13, GH18, and PL1) were identified, and proteases, oxidases, reductases, phospholipases, transferases, and proteins with unknown functions were also detected.

Comparative secretome analysis of rat stomach under different nutritional status.

The fact that gastric surgery is at the moment the most effective treatment to fight against obesity highlights the relevance of gastric derived proteins as potential targets to treat this pathology. Taking advantage of a previously established gastric explant model for endocrine studies, the proteomic analysis of gastric secretome was performed. To validate this gastric explant system for proteomic analysis, the identification of ghrelin, a classical gastric derived peptide, was performed by MS. In addition, the differential analysis of gastric secretomes under differential nutritional status (control feeding vs fasting vs re-feeding) was performed. The MS identified proteins are showed in the present manuscript. The data supplied in this article is related to the research article entitled “Comparative secretome analysis of rat stomach under different nutritional status” [1].

Secretome analysis revealed adaptive and non-adaptive responses of the Staphylococcus carnosus femB mutant.

FemABX peptidyl transferases are involved in non-ribosomal pentaglycine interpeptide bridge biosynthesis. Here, we characterized the phenotype of a Staphylococcus carnosus femB deletion mutant, which was affected in growth and showed pleiotropic effects such as enhanced methicillin sensitivity, lysostaphin resistance, cell clustering, and decreased peptidoglycan cross-linking. However, comparative secretome analysis revealed a most striking difference in the massive secretion or release of proteins into the culture supernatant in the femB mutant than the wild type. The secreted proteins can be categorized into typical cytosolic proteins and various murein hydrolases. As the transcription of the murein hydrolase genes was up-regulated in the mutant, they most likely represent an adaption response to the life threatening mutation. Even though the transcription of the cytosolic protein genes was unaltered, their high abundance in the supernatant of the mutant is most likely due to membrane leakage triggered by the weakened murein sacculus and enhanced autolysins.

On-Site Enzyme Production by Trichoderma asperellum for the Degradation of Duckweed

The on-site production of cell wall degrading enzymes is an important strategy for the development of sustainable bio-refinery processes. This study concerns the optimization of production of plant cell wall-degrading enzymes produced by Trichoderma asperellum. A comparative secretome analysis was performed on T. asperellum growing on PDA agar, wheat bran and duckweed, respectively. T. asperellum proved to be able to produce a wide enzyme profile, including both depolymerization and debranching enzymes, mainly consisting of hemi-cellulases. The secretome analysis showed specific glycoside hydrolase-induction on duckweed compared with growth on wheat bran and PDA, including a GH62 α-L-arabinofuranosidase, a promising candidate enzyme for the degradation of duckweed. The enzyme cocktail from T. asperellum proved capable of degrading pretreated duckweed, obtaining up to 60% of the theoretical glucose yield, making it a potential candidate for on-site enzyme production.

Role of VcrD1 protein in expression and secretion of flagellar components in Vibrio parahaemolyticus.

VcrD1 protein is a component of type III secretion system (T3SS) 1 in Vibrio parahaemolyticus. A comparative analysis of secretomes of wild-type and ΔvcrD1 strains revealed that the mutant was defective in secretion of diverse proteins including several flagellar components. Western blot analyses using specific antibodies confirmed that the secretion of at least four flagellar components, such as FlaA, FlgL, FlgE, and FlgM, was affected by the vcrD1 mutation, which was consistent with decreased motility on soft agar plates and the non-flagellated morphology of the mutant. The ΔexsA mutant, another T3SS1 mutant, did not showed reduced motility, but became non-motile phenotype with the additional ΔvcrD1 mutation. Complementation of wild-type vcrD1 gene into ΔvcrD1 mutant resulted in restored motility. Fractionation of bacterial cytoplasm from the periplasm and membrane revealed lower levels of FlaA and FlgM in the cytoplasm of the ΔvcrD1 mutant, indicating that VcrD1 might regulate the expression of flagellar genes in addition to the secretion of flagellar components in V. parahaemolyticus. Quantitative RT-PCR assays of seven representative flagellar genes in the wild-type and ΔvcrD1 mutant strains demonstrated that transcript levels of two early flagellar genes, flaK and flaL, were not reduced by the vcrD1 mutation, whereas the middle and late flagellar genes were expressed at a lower level in the vcrD1 mutant. This study raises a possibility that VcrD1 plays a role in flagellar morphogenesis in V. parahaemolyticus by regulating the expression and secretion of flagellar components.

Circulating pEGFR is a candidate response biomarker of cetuximab therapy in colorectal cancer.

The lack of secreted biomarkers measurable by noninvasive tests hampers the development of effective targeted therapies against cancer. Our hypothesis is that cetuximab (an anti-EGFR mAb) induces a specific secretome in colorectal cancer cells that could be exploited for biomarker discovery. Considering the strong correlation between mutated KRAS and a lack of response to cetuximab therapy, we addressed whether performing secretome-based proteomics on isogenic colorectal cancer cells sharing the KRAS mutations found on patients would yield candidate-secreted biomarkers useful in the clinical setting. Because 2D culture did not optimally model the sensitivity/resistance to cetuximab observed in colorectal cancer patients, we moved to 3D spheroids, developing a methodology for both cell-based assays and quantitative proteomics. A large comparative quantitative proteomic analysis of the 3D secretomes of colorectal cancer isogenic cells treated with cetuximab uncovered an EGFR pathway-centric secretome found only when cells grow in 3D. The validation of the secretome findings in plasma of colorectal cancer patients, suggests that phosphorylated-EGFR (pEGFR) is a candidate-secreted biomarker of response to cetuximab. We have proved that 3D spheroids from colorectal cancer cells generate secretomes with a drug-sensitivity profile that correlates well with patients with colorectal cancer, illustrating molecular connections between intracellular and extracellular signaling. Furthermore, we show how the secretion of pEGFR is associated with the sensitivity of colorectal cancer cells to cetuximab and the response of patients with colorectal cancer to the drug. Our work could allow the noninvasive monitoring of anti-EGFR treatment in patients with colorectal cancer.

Comparative secretome analysis of cholangiocarcinoma cell line in three-dimensional culture.

Cholangiocarcinoma (CCA) is a lethal malignancy which occurs with relatively high incidence in Thailand. This cancer is often difficult to diagnose and associated with high mortality. The secretome, containing the secreted proteins from cells, are potentially useful as biomarkers of cancers. Since three-dimensional (3D) cell culture may mimic growth characteristics and microenvironment of solid tumors in vivo better than monolayer culture, we have developed culture of CCA in natural collagen-based scaffold, to enable analysis of the secretome by 2DE. Our results indicated that CCA growth in 3D environment alters cell shape significantly and enhances extracellular matrix deposition. Interestingly, more secreted proteins were detected from 3D culture compared to monolayer culture. Secretome analysis using 2DE coupled with LC-MS/MS demonstrated 10 secreted proteins uniquely found in 3D culture. Moreover, 25 proteins were enriched in 3D culture compared to monolayer culture, including 14-3-3 σ, triosephosphate isomerase, phosphoglycerate mutase 1, α-enolase, and L-plastin. Immunoblotting was used to confirm the presence of L-plastin in conditioned media of CCA and of hepatocellular carcinoma (HCC) cell lines. The results revealed that L-plastin, an actin bundling protein, was uniquely expressed only in the CCA cell line and could be a promising biomarker for differential diagnosis of CCA compared to HCC.

Comparative secretome analysis of Streptomyces scabiei during growth in the presence or absence of potato suberin.

BackgroundSuberin is a recalcitrant plant biopolymer composed of a polyphenolic and a polyaliphatic domain. Although suberin contributes to a significant portion of soil organic matter, the biological process of suberin degradation is poorly characterized. It has been suggested that Streptomyces scabiei, a plant pathogenic bacterium, can produce suberin-degrading enzymes. In this study, a comparative analysis of the S. scabiei secretome from culture media supplemented or not with potato suberin was carried out to identify enzymes that could be involved in suberin degradation.MethodsS. scabiei was grown in the presence of casein only or in the presence of both casein and suberin. Extracellular proteins from 1-, 3- and 5-day-old supernatants were analyzed by LC-MS/MS to determine their putative functions. Real-time RT-PCR was performed to monitor the expression level of genes encoding several proteins potentially involved in suberin degradation.ResultsThe effect of suberin on the extracellular protein profile of S. scabiei strain has been analyzed. A total of 246 proteins were found to be common in the data sets from both casein medium (CM) and casein-suberin medium (CSM), whereas 124 and 139 proteins were detected only in CM or CSM, respectively. The identified proteins could be divided into 19 functional groups. Two functional groups of proteins (degradation of aromatic compounds and secondary metabolism) were only associated with the CSM. A high proportion of the proteins found to be either exclusively produced, or overproduced, in presence of suberin were involved in carbohydrate metabolism. Most of the proteins included in the lipid metabolism class have been detected in CSM. Apart from lipid metabolism proteins, other identified proteins, particularly two feruloyl esterases, may also actively participate in the breakdown of suberin architecture. Both feruloyl esterase genes were overexpressed between 30 to 340 times in the presence of suberin.ConclusionThis study demonstrated that the presence of suberin in S. scabiei growth medium induced the production of a wide variety of glycosyl hydrolases. Furthermore, this study has allowed the identification of extracellular enzymes that could be involved in the degradation of suberin, including enzymes of the lipid metabolism and feruloyl esterases.

Comparative analysis of secretomes in basidiomycete fungi

Lignocellulose, the major reservoir of organic carbon on Earth, is recalcitrant to turnover and resistant to microbial and enzymatic attack because of the protective action of lignin and the crystalline structure of cellulose. Basidiomycetes are the only organisms known to degrade lignocellulose using two alternative degradation strategies: white rot basidiomycetes break down the lignin moiety extensively before attacking the cellulose, whereas brown rot basidiomycetes cause limited lignin alteration while primarily degrading cellulose. These strategies depend on complex portfolios of extracellular enzymes whose production and export are environment and substrate dependent. More than 265 fungal genomes (more than 90 corresponding to basidiomycetes) have been sequenced. These sequences can be bioinformatically screened to identify presumptive secreted proteins that, however, differ from the experimental data, stressing the importance of using complementary approaches for analyzing these complex systems. The study of fungal secretomes from the scope of their different lignin degradation strategies and lifestyles would facilitate their use in the treatment of lignocellulose as carbon feedstock for biofuel production and further biorefinery processes. In this article, we carry out a comparative review of the analytical tools and strategies used for studying basidiomycetes' secretomes and compare the available results from these two points of view. In this article, we review the tools and strategies used in the analysis of basidiomycetes' secretomes and we compare the secretome data available for this type of fungi with a special emphasis on the enzymes involved in lignocellulose degradation. This comparative study provides a basis for distinguishing different lifestyles (saprotrophs, symbionts and parasites) on the basis of their respective secretomes.

Comparative Secretome Analysis of Epithelial and Mesenchymal Subpopulations of Head and Neck Squamous Cell Carcinoma Identifies S100A4 as a Potential Therapeutic Target

Epithelial-mesenchymal transition (EMT) is a key contributor in tumor progression and metastasis. EMT produces cellular heterogeneity within head and neck squamous cell carcinomas (HNSCC) by creating a phenotypically distinct mesenchymal subpopulation that is resistant to conventional therapies. In this study, we systematically characterized differences in the secretomes of E-cadherin high epithelial-like and E-cadherin low mesenchymal-like subpopulations using unbiased and targeted proteomics. A total 1765 proteins showed significant changes with 177 elevated in the epithelial subpopulation and 173 elevated in the mesenchymal cells. Key nodes in affected networks included NFκB, Akt, and ERK, and most implicated cellular components involved various aspects of the extracellular matrix. In particular, large changes were observed in multiple collagens with most affected collagens at much higher abundance levels in the mesenchymal subpopulation. These cells also exhibited a secretome profile resembling that of cancer-associated fibroblastic cells (CAF). S100A4, a commonly used marker for cancer-associated fibroblastic cells, was elevated more than 20-fold in the mesenchymal cells and this increase was further verified at the transcriptome level. S100A4 is a known mediator of EMT, leading to metastasis and EMT has been proposed as a potential source of cancer-associated fibroblastic cells in solid tumors. S100A4 knockdown by small interfering RNA led to decreased expression, secretion and activity of matrix metalloproteinase 2, as verified by quantitative PCR, multiple reaction monitoring and zymography analyses, and reduced invasion in collagen-embedded spheroids. Further confirmation in three-dimensional organotypic reconstructs showed less invasion and advanced differentiation in the S100A4 RNA interference samples. Orthotopic metastasis model, developed to validate the findings in vivo, demonstrated a decrease in spontaneous metastasis and augmented differentiation in the primary tumor in siS100A4 xenografts. These results demonstrate the value of secretome profiling to evaluate phenotypic conversion and identify potential novel therapeutic targets such as S100A4.

Comparative secretome analysis of Trichoderma asperellum S4F8 and Trichoderma reesei Rut C30 during solid-state fermentation on sugarcane bagasse.

BackgroundThe lignocellulosic enzymes of Trichoderma species have received particular attention with regard to biomass conversion to biofuels, but the production cost of these enzymes remains a significant hurdle for their commercial application. In this study, we quantitatively compared the lignocellulolytic enzyme profile of a newly isolated Trichoderma asperellum S4F8 strain with that of Trichoderma reesei Rut C30, cultured on sugarcane bagasse (SCB) using solid-state fermentation (SSF).ResultsComparison of the lignocellulolytic enzyme profiles of S4F8 and Rut C30 showed that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities. Liquid chromatography tandem mass spectrometry analysis of the two fungal secretomes enabled the detection of 815 proteins in total, with 418 and 397 proteins being specific for S4F8 and Rut C30, respectively, and 174 proteins being common to both strains. In-depth analysis of the associated biological functions and the representation of glycoside hydrolase family members within the two secretomes indicated that the S4F8 secretome contained a higher diversity of main and side chain hemicellulases and β-glucosidases, and an increased abundance of some of these proteins compared with the Rut C30 secretome.ConclusionsIn SCB SSF, T. asperellum S4F8 produced a more complex lignocellulolytic cocktail, with enhanced hemicellulose and cellobiose hydrolysis potential, compared with T. reesei Rut C30. This bodes well for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail from T. asperellum for lignocellulosic feedstock hydrolysis.

Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay

To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (LPMO, formerly GH61), also have been implicated in cellulose degradation. To examine polysaccharide-degrading potential between white- and brown-rot fungi, we performed genomewide analysis of CAZys and these oxidative enzymes in 11 Polyporales, including recently sequenced monokaryotic strains of Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora. Furthermore, we conducted comparative secretome analysis of seven Polyporales grown on wood culture. As a result, it was found that genes encoding cellulases belonging to families GH6, GH7, GH9 and carbohydrate-binding module family CBM1 are lacking in genomes of brown-rot polyporales. In addition, the presence of CDH and the expansion of LPMO were observed only in white-rot genomes. Indeed, GH6, GH7, CDH and LPMO peptides were identified only in white-rot polypores. Genes encoding aldose 1-epimerase (ALE), previously detected with CDH and cellulases in the culture filtrates, also were identified in white-rot genomes, suggesting a physiological connection between ALE, CDH, cellulase and possibly LPMO. For hemicellulose degradation, genes and peptides corresponding to GH74 xyloglucanase, GH10 endo-xylanase, GH79 β-glucuronidase, CE1 acetyl xylan esterase and CE15 glucuronoyl methylesterase were significantly increased in white-rot genomes compared to brown-rot genomes. Overall, relative to brown-rot Polyporales, white-rot Polyporales maintain greater enzymatic diversity supporting lignocellulose attack.

Comparative secretome analysis ofFusarium graminearumand two of its non-pathogenic mutants upon deoxynivalenol induction in vitro

To understand early events in plant-pathogen interactions, it is necessary to explore the pathogen secretome to identify secreted proteins that help orchestrate pathology. The secretome can be obtained from pathogens grown in vitro, and then characterized using standard proteomic approaches based on protein extraction and subsequent identification of tryptic peptides by LC-MS. A subset of the secretome is composed of proteins whose presence is required to initiate infection and their removal from the secretome would result in pathogens with reduced or no virulence. We present here comparative secretome from Fusarium graminearum. This filamentous fungus causes Fusarium head blight on wheat, a serious cereal disease found in many cereal-growing regions. Affected grain is contaminated with mycotoxins and cannot be used for food or feed. We used label-free quantitative MS to compare the secretomes of wild-type with two nonpathogenic deletion mutants of F. graminearum, Δtri6, and Δtri10. These mutations in mycotoxin-regulating transcription factors revealed a subset of 29 proteins whose relative abundance was affected in their secretomes, as measured by spectral counting. Proteins that decreased in abundance are potential candidate virulence factors and these included cell wall-degrading enzymes, metabolic enzymes, pathogenesis-related proteins, and proteins of unknown function.

Abstract 2510: Comparative proteomic analysis of melanoma cell secretomes reveals autophagy-related biomarkers.

Abstract Autophagy is a highly conserved mechanism of cellular self-digestion that serves as a major protein and organelle degrading pathway and a critical adaptive response to nutrient, endoplasmic reticulum, and oxidative stress. Autophagy has been implicated as a survival mechanism in melanoma tumorigenesis and has been recognized as a primary mechanism of chemotherapeutic resistance. Pre-treatment analyses of autophagy levels in melanoma tumor samples have shown a high degree of heterogeneity across patients, with high levels of autophagy correlating with significantly shorter survival. However, quantitative assays for autophagy remain elusive at the pre-clinical level, impeding further understanding as to its role in melanoma progression. Another challenge to developing effective markers of autophagy is the striking difference in autophagic response of cells grown in conventional monolayer culture versus cells growing in the tumor microenvironment. Recent studies have found that autophagy regulates ‘unconventional’ protein secretion pathways, resulting in secretion of a subset of cytosolic proteins. Therefore, the focus of this work was to investigate molecular changes in the melanoma cell secretome as a means of identifying key proteins diagnostic of autophagy levels. To approximate autophagy dynamics in the tumor microenvironment, human melanoma cells were grown as spheroids in Type I bovine collagen matrices. To enrich for autophagy-related secretions, secretomes of low-autophagy WM793 cells and the genetically closely related high-autophagy 1205Lu cells were directly compared at baseline. Conditioned media was collected and separated using 1-D gel electrophoresis, digested with trypsin, and analyzed using liquid chromatography-tandem mass spectrometry analysis on an LTQ-Orbitrap hybrid instrument. Mass spectra were analyzed using the Rosetta ELUCIDATOR proteomic pipeline, and significance analysis of microarrays (SAM) was used to identify differentially secreted proteins. From Gene Ontology (GO) analysis, candidate biomarkers were enriched for extracellular matrix components, inflammatory mediators, and metabolic enzymes implicated in autophagy and melanoma progression. A subset of these proteins was predicted to be secreted via a non-classical mechanism using SecretomeP and SignalP secretory protein prediction servers. Network analysis using the shortest paths algorithm identified densely connected sub-networks that illustrated interplay between inflammatory cytokines and metastasis-related proteins, providing insight into molecular pathways that may regulate constitutively elevated autophagy. Our findings indicate that differences in basal autophagy in human melanoma manifest as secretory changes that could be novel serological biomarkers of autophagy for cancer prognosis and therapy. Citation Format: Adam Akl, Ravi Amaravadi, David Speicher. Comparative proteomic analysis of melanoma cell secretomes reveals autophagy-related biomarkers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2510. doi:10.1158/1538-7445.AM2013-2510

Comparative secretome analysis of four isogenic Bacillus clausii probiotic strains

BackgroundThe spore-bearing alkaliphilic Bacillus species constitute a large, heterogeneous group of microorganisms, important for their ability to produce enzymes, antibodies and metabolites of potential medical use. Some Bacillus species are currently being used for manufacturing probiotic products consisting of bacterial spores, exhibiting specific features (colonization, immune-stimulation and antimicrobial activity) that can account for their claimed probiotic properties. In the present work a comparative proteomic study was performed aimed at characterizing the secretome of four closely related isogenic O/C, SIN, N/R and T B. clausii strains, already marketed in a pharmaceutical mixture as probiotics.ResultsProteomic analyses revealed a high degree of concordance among the four secretomes, although some proteins exhibited considerable variations in their expression level in the four strains. Among these, some proteins with documented activity in the interaction with host cells were identified, such as the glycolytic enzyme enolase, with a putative plasminogen-binding activity, GroEL, a molecular chaperone shown to be able to bind to mucin, and flagellin protein, a structural flagella protein and a putative immunomodulation agent.ConclusionThis study shows, for the first time, differences in the secretome of the OC, SIN, NR and T B. clausii strains. These differences indicate that specific secretome features characterize each of the four strains despite their genotypic similarity. This could confer to the B. clausii strains specific probiotic functions associated with the differentially expressed proteins and indicate that they can cooperate as probiotics as the secretome components of each strain could contribute to the overall activity of a mixed probiotic preparation.

Comparative Secretome Analyses Using a Hollow Fiber Culture System with Label-Free Quantitative Proteomics Indicates the Influence of PARK7 on Cell Proliferation and Migration/Invasion in Lung Adenocarcinoma

As the leading cause of cancer death worldwide, lung cancer lacks effective diagnosis tools and treatments to prevent its metastasis. Fortunately, secretome has clinical usages as biomarkers and protein drugs. To discover the secretome that influences lung adenocarcinoma metastasis, the hollow fiber culture (HFC) system was used along with label-free proteomics approach to analyze cell secretomes between CL1-0 and CL1-5 cell lines, which exhibit low and high metastatic potentials. Among the 703 proteins quantified, 50 possessed different levels between CL1-0 and CL1-5. PARK7 was a primary focus because of the lack of research involving lung adenocarcinoma. The cell proliferation, migration, and invasion properties of CL1-0, CL1-5, and A549 cells were significantly diminished when the expression of their PARK7 proteins was reduced. Conversely, these functions were promoted when PARK7 was overexpressed in CL1-0. In clinical expression, PARK7 levels within tissue specimens and plasma samples were significantly higher in the cancer group. This represents the first time the HFC system has been used with label-free quantification to discern the elements of metastasis in lung adenocarcinoma cell secretomes. Likewise, PARK7 has never been researched for its role in promoting lung adenocarcinoma progression.

Comparative secretome analysis of human bone marrow‐derived mesenchymal stem cells during osteogenesis

Osteogenesis is a tightly regulated process that involves coordinated extracellular signals from autocrine and paracrine loops. Secretory proteins during osteogenesis can inhibit cell proliferation and activate cell differentiation toward mature osteoblasts, which are characterized by mineralization. In this study, we attempted to identify these secretory proteins during osteogenesis using LC-MS/MS analysis. We compared the secretome between undifferentiated human bone marrow-derived mesenchymal stem cells (hBMSCs) and differentiated osteoblasts. Among 315 proteins that were identified, 177 proteins were present at increased levels in osteoblasts, whereas 88 proteins were present at decreased levels. Among the identified proteins, several were validated by quantitative RT-PCR and immunoblot analysis. Of particular interest, calcium homeostasis-related proteins were upregulated, whereas stem cell proliferation-related proteins and other lineage-related proteins were downregulated during osteogenesis. These findings provide information about the dynamic changes in the expression and secretion of proteins during osteogenesis and suggest the putative role of secretory proteins in osteogenesis.

Comparative secretome analysis of human follicular dermal papilla cells and fibroblasts using shotgun proteomics

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically- validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.