Changes In Secretome Research Articles

Proteomic Analysis of the Secretory Response of Aspergillus niger to D-Maltose and D-Xylose

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on d-sorbitol, small amounts of d-maltose or d-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by d-maltose or d-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on d-maltose and β-xylosidase D on d-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra d-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of d-xylose or d-maltose. Furthermore, d-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers d-maltose and d-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by d-maltose or d-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for d-xylose induction, d-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.

Abstract 435: MT regulation of tumor-dependent microenvironment remodeling

Abstract Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, drugs that target MTs, like the taxanes, often fail in the metastatic setting. Most studies are performed in a two-dimensional monoculture thus, overlook the complexity of the tumor microenvironment. Therefore, to gain a better understanding of tumor MT biology within a physiological context we studied the role of MT dynamics in 3D co-cultures closer mimicking the tumor microenvironment. Since tumor cells need to remodel their ECM in order to metastasize we hypothesized that the interphase MT cytoskeleton, through the coordination of cellular signaling regulates this process. To test this hypothesis we first assessed the MT-dependence of tumor-mediated ECM remodeling, using a floating collagen gel contraction assay. In this assay, breast cancer cells were embedded into a floating collagen gel and monitored for their ability to contract the gel as a result of cell generated traction force. We observed that the MDA-MB-231 metastatic breast cancer cells exhibited greater contractility as compared with the MCF-7 non-metastatic breast cancer cells or the non-tumorigenic human breast epithelial cell line MCF10A. However upon disruption of MTs with paclitaxel (PTX), tumor cell contractility was severely inhibited at drug concentrations that did not affect cell number, suggesting that dynamic MTs are essential for cell traction force generation. In addition, we observed that human mammary fibroblasts when co-cultured with breast cancer cells in 3D exhibited increased motility as compared to 3D fibroblast monocultures. Interestingly, fibroblast activation was inhibited in co-cultures with breast cancer cells previously treated with a taxane, suggesting that tumor MT stabilization negatively affects fibroblast activation. To investigate the factors that mediated the tumor cell communication with surrounding fibroblasts we monitored fibroblast motility in 3D monocultures following incubation with conditioned medium collected from either untreated or Taxol pre-treated breast cancer cells. In agreement with our 3D co-culture results, fibroblast 3D motility was inhibited following exposure to conditioned medium from the Taxol-pretreated cells. These results suggest that the composition of the tumor secretome changes upon MT stabilization, which in turn affects the ability of tumor cells to communicate with stromal fibroblasts. Currently, we are performing proteomic analysis of the conditioned media to identify soluble factors whose secretion is dependent on dynamic microtubules. In addition, to determine whether the effects of taxane treatment is solely due to microtubule stabilization we are performing the 3D co-culture experiments using a Taxol-resistant β-tubulin mutant breast cancer cell line. These findings point to a novel role of interphase MTs in tumor biology and can possibly lead to new avenues for taxane clinical efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 435. doi:10.1158/1538-7445.AM2011-435

Platelet Microparticles Enhance the Vasoregenerative Potential of Angiogenic Early Outgrowth Cells After Vascular Injury

Angiogenic early outgrowth cells (EOCs) have been reported to contribute to endothelial regeneration and to limit neointima formation after vascular injury. Vascular pathologies comprise platelet activation and concomitant generation of platelet microparticles (PMPs). We hypothesized that PMPs may interact with EOCs in the context of vascular injury and modulate their regenerative potential. Using flow cytometry, confocal microscopy, and scanning electron microscopy, we demonstrated the binding of thrombin/collagen-induced PMPs to EOCs with subsequent membrane assimilation and incorporation. This interaction promoted phenotypic alterations of EOCs with increased expression of endothelial cell markers and transfer of the chemokine receptor CXCR4 to EOCs with enhanced responsiveness to its ligand CXCL12/SDF-1alpha. In addition, PMPs augmented the adhesion of EOCs to extracellular matrix components and to the injured vessel wall and accelerated cytoskeletal reorganization and migration of EOCs. PMPs induced changes in the EOC secretome toward a more proangiogenic profile and amplified the EOC-mediated induction of proliferation, migration, and capillary tube formation by mature endothelial cells. Compared with untreated EOCs, the injection of PMP-treated EOCs resulted in accelerated reendothelialization after arterial denudation injury in athymic nude mice, whereas the EOC-mediated reduction of neointima formation remained unchanged. Our data provide evidence that PMPs can boost the potential of EOCs to restore endothelial integrity after vascular injury. Major mechanisms involve the enhancement of EOC recruitment, migration, differentiation, and release of proangiogenic factors.

The influence of inflammation on the human visceral adipose tissue secretome

Chronic inflammation of obese adipose tissue is involved in the development of insulin resistance and type 2 diabetes, probably via altered secretion of adipokines and free fatty acids. To determine how inflammation changes the adipose tissue secretome, adipose tissue was challenged with LPS, a potent immune activator. Secretome changes were analyzed by a quantitative proteomics approach: Comparison of Isotope Labeled Amino acid Incorporation Rates (CILAIR). CILAIR compares incorporation rates of 13C‐labeled lysine in secreted proteins between conditions (Mol Cell Proteomics 2009, 8:316).MethodsHuman visceral adipose tissue was divided over six dishes. 13C‐Lys containing medium was added and to three dishes also LPS (100 μg/ml). Dishes were incubated for 24 hrs. Media were collected and processed separately, involving SDS‐PAGE fractionation, in‐gel digestion of excised bands and LC‐MS/MS. Database searching with ProteinPilot (Applied Biosystems) provided identifications and C13/C12 ratios.Results37 proteins were significantly changed (p<0.05) in expression of which 19 were up‐regulated by LPS and 19 were down‐regulated. Examples of up‐regulated proteins are: Plasminogen activator inhibitor 2, Growth‐regulated alpha protein and Interleukin‐6. In addition, 12 proteins were only expressed in the inflammatory condition. Examples are: Tumor necrosis factor‐α and C‐C motif chemokine 2 and 20.ConclusionThese results indicate that inflammation profoundly changes the adipose tissue secretome. Secretion of several inflammatory cytokines and chemokines was up‐regulated as well as proteins involved in cell adhesion of attracted immune cells. These findings may have implications for the regulation of whole body insulin sensitivity and energy metabolism.

Growth‐related variations in the Bacillus cereus secretome

Using 2-DE, transcriptional gene fusions and cell cytotoxicity assays, we followed changes in the Bacillus cereus strain ATCC14579 secretome, gene expression and culture supernatant cytotoxicity from the end of the vegetative phase up to 5 h after entry into the stationary phase. The concentration of each of the 22 proteins in the culture supernatant was determined at various times. In addition, the stability of the proteins was studied. Fifteen of these proteins, including 14 members of the virulence regulon PlcR, were known or predicted to be secreted. All of the secreted proteins reached a maximum concentration during early stationary phase, but there were significant differences in the kinetics of their concentrations. The time courses of protein concentrations were in agreement with gene expression data, except for cytotoxin CytK, which was unstable, and for the metalloprotease InhA1. Supernatant cytoxicity also peaked in early stationary phase, and the kinetics of cytotoxicity paralleled the time course of concentration of the PlcR-controlled toxin, CytK. Our concomitant study of the time course of protein concentrations, gene expression and supernatant cytotoxicity reveals that the pathogenic potential of B. cereus peaks during the transition state. It also suggests that there is diversity in the regulation of gene expression within the PlcR regulon.