Modulation of bone marrow mesenchymal stem cell secretome by ECM-like hydrogels

It has been demonstrated that bone marrow mesenchymal stem cell (BM-MSCs) transplantation has beneficial effects on several central nervous system (CNS) debilitating conditions. Growing evidence indicate that trophic factors secreted by these cells are the key mechanism by which they are acting. These cells are frequently used in combination with 3D artificial matrices, for instance hydrogels, in tissue engineering-based approaches. However, so far, no study has been reported on the influence of such matrices, namely the presence or absence of extracellular matrix motifs, on BM-MSCs secretome and its effects in neuronal cell populations. In this sense, we herein studied the impact of a hydrogel, gellan gum, on the behavior and secretome of BM-MSCs, both in its commercial available form (commonly used in tissue engineering) and in a fibronectin peptide-modified form. The results showed that in the presence of a peptide in the gellan gum hydrogel, BM-MSCs presented higher proliferation and metabolic activity than in the regular hydrogel. Moreover, the typical spindle shape morphology of BM-MSCs was only observed in the modified hydrogel. The effects of the secretome of BM-MSCs were also affected by the chemical nature of the extracellular matrix. BM-MSCs cultured in the modified hydrogel were able to secrete factors that induced higher metabolic viabilities and neuronal cell densities, when compared to those of the unmodified hydrogel. Thus adding a peptide sequence to the gellan gum had a significant effect on the morphology, activity, proliferation and secretome of BM-MSCs. These results highlight the importance of mimicking the extracellular matrix when BM-MSCs are cultured in hydrogels for CNS applications.     

Shotgun Analysis of the Secretome of Fusarium graminearum

Fusarium head blight, caused predominately by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. To characterize the profile of proteins secreted by F. graminearum, the extracellular proteins were collectively obtained from F. graminearum culture supernatants and evaluated using one-dimensional SDS-PAGE and liquid chromatography-tandem mass spectrometry. A total of 87 proteins have been identified, of which 63 were predicted as secretory proteins including those with known functions. Meanwhile, 20 proteins that are not homologous to genomic sequences with known functions have also been detected. Some of the identified proteins are possible virulence factors and may play extracellular roles during F. graminearum infection. This study provides a valuable dataset of F. graminearum extracellular proteins, and a better understanding of the virulence mechanisms of the pathogen.     

The mesenchymal stem cell secretome: A new paradigm towards cell-free therapeutic mode in regenerative medicine

Mesenchymal Stem Cells (MSCs) have been shown to be a promising candidate for cell-based therapy. The therapeutic potential of MSCs, towards tissue repair and wound healing is essentially based on their paracrine effects. Numerous pre-clinical and clinical studies of MSCs have yielded encouraging results. Further, these cells have been shown to be relatively safe for clinical applications. MSCs harvested from numerous anatomical locations including the bone marrow, adipose tissue, Wharton’s jelly of the umbilical cord etc., display similar immunophenotypic profiles. However, there is a large body of evidence showing that MSCs secrete a variety of biologically active molecules such as growth factors, chemokines, and cytokines. Despite the similarity in their immunophenotype, the secretome of MSCs appears to vary significantly, depending on the age of the host and niches where the cells reside. Thus, by implication, proteomics-based profiling suggests that the therapeutic potential of the different MSC populations must also be different. Analysis of the secretome points to its influence on varied biological processes such as angiogenesis, neurogenesis, tissue repair, immunomodulation, wound healing, anti-fibrotic and anti-tumour for tissue maintenance and regeneration. Though MSC based therapy has been shown to be relatively safe, from a clinical standpoint, the use of cell-free infusions can altogether circumvent the administration of viable cells for therapy. Understanding the secretome of in vitro cultured MSC populations, by the analysis of the corresponding conditioned medium, will enable us to evaluate its utility as a new therapeutic option. This review will focus on the accumulating evidence that points to the therapeutic potential of the conditioned medium, both from pre-clinical and clinical studies. Finally, this review will emphasize the importance of profiling the conditioned medium for assessing its potential for cell-free therapy therapy.     

Assessing proteolytic events in bioinformatic reanalysis of public secretome data from melanoma cell lines

Autocrine and paracrine signals are of paramount importance in both normal and oncogenic events and the composition of such secreted molecular signals (i.e the secretome) designate the communication status of cells. In this context, the analysis of post-translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as proteolytic processing. In the present study, we have performed a bioinformatic reanalysis of public proteomics data on melanoma cell line secretomes, changing database searching parameters to allow for the identification of proteolytic events generated by active proteases. Such approach enabled the identification of proteolytic signatures which suggested active proteases and whose expression profiles might be targeted in patient tissues or liquid biopsies, as well as their cleaved substrates. Although N-terminomics approaches continue to be the method of choice for the evaluation of proteolytic signaling events in complex samples, the simple approach performed in this work resulted in the gain of biological insights derived from shotgun proteomics data.     

Methodologies to decipher the cell secretome

The cell secretome is a collection of proteins consisting of transmembrane proteins (TM) and proteins secreted by cells into the extracellular space. A significant portion (~ 13–20%) of the human proteome consists of secretory proteins. The secretory proteins play important roles in cell migration, cell signaling and communication. There is a plethora of methodologies available like Serial Analysis of Gene Expression (SAGE), DNA microarrays, antibody arrays and bead-based arrays, mass spectrometry, RNA sequencing and yeast, bacterial and mammalian secretion traps to identify the cell secretomes. There are many advantages and disadvantages in using any of the above methods. This review aims to discuss the methodologies available along with their potential advantages and disadvantages to identify secretory proteins. This review is a part of a Special issue on The Secretome. This article is part of a Special Issue entitled: An Updated Secretome.     

Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation

Background

Since the first fungal genome sequences became available, investigators have been employing comparative genomics to understand how fungi have evolved to occupy diverse ecological niches. The secretome, i.e. the entirety of all proteins secreted by an organism, is of particular importance, as by these proteins fungi acquire nutrients and communicate with their surroundings.

Results

It is generally assumed that fungi with similar nutritional lifestyles have similar secretome compositions. In this study, we test this hypothesis by annotating and comparing the soluble secretomes, defined as the sets of proteins containing classical signal peptides but lacking transmembrane domains of fungi representing a broad diversity of nutritional lifestyles. Secretome size correlates with phylogeny and to a lesser extent with lifestyle. Plant pathogens and saprophytes have larger secretomes than animal pathogens. Small secreted cysteine-rich proteins (SSCPs), which may comprise many effectors important for the interaction of plant pathogens with their hosts, are defined here to have a mature length of ≤ 300 aa residues, at least four cysteines, and a total cysteine content of ≥5%. SSCPs are found enriched in the secretomes of the Pezizomycotina and Basidiomycota in comparison to Saccharomycotina. Relative SSCP content is noticeably higher in plant pathogens than in animal pathogens, while saprophytes were in between and closer to plant pathogens. Expansions and contractions of gene families and in the number of occurrences of functional domains are largely lineage specific, e.g. contraction of glycoside hydrolases in Saccharomycotina, and are only weakly correlated with lifestyle. However, within a given lifestyle a few general trends exist, such as the expansion of secreted family M14 metallopeptidases and chitin-binding proteins in plant pathogenic Pezizomycotina.

Conclusions

While the secretomes of fungi with similar lifestyles share certain characteristics, the expansion and contraction of gene families is largely lineage specific, and not shared among all fungi of a given lifestyle.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-722) contains supplementary material, which is available to authorized users.     

Identification of proteins secreted by head and neck cancer cell lines using LC-MS/MS: Strategy for discovery of candidate serological biomarkers

In search of blood-based biomarkers that would enhance the ability to diagnose head and neck/oral squamous cell carcinoma (HNOSCC) in early stages or predict its prognosis, we analyzed the HNOSCC secretome (ensemble of proteins secreted and/or shed from the tumor cells) for potential biomarkers using proteomic technologies. LC-MS/MS was used to identify proteins in the conditioned media of four HNOSCC cell lines (SCC4, HSC2, SCC38, and AMOSIII); 140 unique proteins were identified on the basis of 5% global false discovery rate, 122 of which were secretory proteins, with 29 being previously reported to be overexpressed in HNOSCC in comparison to normal head and neck tissues. Of these, five proteins including α-enolase, peptidyl prolyl isomerase A/cyclophilin A, 14-3-3 ζ, heterogeneous ribonucleoprotein K, and 14-3-3 σ were detected in the sera of HNOSCC patients by Western blot analysis. Our study provides the evidence that analysis of head and neck cancer cells' secretome is a viable strategy for identifying candidate serological biomarkers for HNOSCC. In future, these biomarkers may be useful in predicting the likelihood of transformation of oral pre-malignant lesions, prognosis of HNOSCC patients and evaluate response to therapy using minimally invasive tests.     

Towards characterization of the glycoproteome of tomato (Solanum lycopersicum) fruit using Concanavalin A lectin affinity chromatography and LC-MALDI-MS/MS analysis

The isolation and analysis of glycoproteins by coupling lectin affinity chromatography with MS has emerged as a powerful strategy to study the glycoproteome of mammalian cells. However, this approach has not been used extensively for the analysis of plant glycoproteins. As with all eukaryotes, N-glycosylation is a common post-translational modification for plant proteins traveling through the secretory pathway. Many such proteins are destined for the cell wall, or apoplast, where they play important roles in processes such as modifying cell wall structure, sugar metabolism, signaling, and defense against pathogens. Here, we describe a strategy to enrich for and identify secreted plant proteins based on affinity chromatography using the lectin Concanavalin A and two-dimensional liquid chromatography, together with matrix-assisted laser desorption/ionization MS analysis. The value of this approach is illustrated through the characterization of glycoproteins that are expressed in ripe tomato (Solanum lycopersicum) fruit, a developmental stage that is fundamentally linked with significant changes in cell wall structure and composition. This glycoprotein trap strategy allowed the isolation of a sub-proteome with an extremely high proportion of proteins that are predicted to be resident in the cell wall or secretory pathway, and the identification of new putative cell wall proteins.     

Secretome analysis of Magnaporthe oryzae using in vitro systems

Magnaporthe oryzae is a devastating blast fungal pathogen of rice (Oryza sativa L.) that causes dramatic decreases in seed yield and quality. During the early stages of infection by this pathogen, the fungal spore senses the rice leaf surface, germinates, and penetrates the cell via an infectious structure known as an appressorium. During this process, M. oryzae secretes several proteins; however, these proteins are largely unknown mainly due to the lack of a suitable method for isolating secreted proteins during germination and appressoria formation. We examined the secretome of M. oryzae by mimicking the early stages of infection in vitro using a glass plate (GP), PVDF membrane, and liquid culture medium (LCM). Microscopic observation of M. oryzae growth revealed appressorium formation on the GP and PVDF membrane resembling natural M. oryzae-rice interactions; however, appresorium formation was not observed in the LCM. Secreted proteins were collected from the GP (3, 8, and 24 h), PVDF membrane (24 h), and LCM (48 h) and identified by two-dimensional gel electrophoresis (2DE) followed by tandem mass spectrometry. The GP, PVDF membrane, and LCM-derived 2D gels showed distinct protein patterns, indicating that they are complementary approaches. Collectively, 53 nonredundant proteins including previously known and novel secreted proteins were identified. Six biological functions were assigned to the proteins, with the predominant functional classes being cell wall modification, reactive oxygen species detoxification, lipid modification, metabolism, and protein modification. The in vitro system using GPs and PVDF membranes applied in this study to survey the M. oryzae secretome, can be used to further our understanding of the early interactions between M. oryzae and rice leaves.