ABSTRACT: Pulldown PMEL NINTA; secretome BXPC3 DMSO vs BACE2 inhibitor (3I), insoluble fractione of secretome in IGR37; insoluble fractione of secretome in BXPC3
Project description:In this inaugural paper, we shall provide an overview of the endothelial surface layer or glycocalyx in several roles: as a transport barrier, as a porous hydrodynamic interface in the motion of red and white cells in microvessels, and as a mechanotransducer of fluid shearing stresses to the actin cortical cytoskeleton of the endothelial cell. These functions will be examined from a new perspective, the quasiperiodic ultrastructural model proposed in Squire et al. [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239-255] for the 3D organization of the endothelial surface layer and its linkage to the submembranous scaffold. We shall show that the core proteins in the bush-like structures comprising the matrix have a flexural rigidity, EI, that is sufficiently stiff to serve as a molecular filter for plasma proteins and as an exquisitely designed transducer of fluid shearing stresses. However, EI is inadequate to prevent the buckling of these protein structures during the intermittent motion of red cells or the penetration of white cell microvilli. In these cellular interactions, the viscous draining resistance of the matrix is essential for preventing adhesive molecular interactions between proteins in the endothelial membrane and circulating cellular components.
Project description:Mammalian cells are covered by a surface proteoglycan (glycocalyx) layer, and it is known that blood vessel-lining endothelial cells use the glycocalyx to sense and transduce the shearing forces of blood flow into intracellular signals. Tumor cells in vivo are exposed to forces from interstitial fluid flow that may affect metastatic potential but are not reproduced by most in vitro cell motility assays. We hypothesized that glycocalyx-mediated mechanotransduction of interstitial flow shear stress is an un-recognized factor that can significantly enhance metastatic cell motility and play a role in augmentation of invasion. Involvement of MMP levels, cell adhesion molecules (CD44, ?3 integrin), and glycocalyx components (heparan sulfate and hyaluronan) was investigated in a cell/collagen gel suspension model designed to mimic the interstitial flow microenvironment. Physiological levels of flow upregulated MMP levels and enhanced the motility of metastatic cells. Blocking the flow-enhanced expression of MMP activity or adhesion molecules (CD44 and integrins) resulted in blocking the flow-enhanced migratory activity. The presence of a glycocalyx-like layer was verified around tumor cells, and the degradation of this layer by hyaluronidase and heparinase blocked the flow-regulated invasion. This study shows for the first time that interstitial flow enhancement of metastatic cell motility can be mediated by the cell surface glycocalyx - a potential target for therapeutics.
Project description:Mammalian cells, including cancer cells, are covered by a surface layer containing cell bound proteoglycans, glycoproteins, associated glycosaminoglycans and bound proteins that is commonly referred to as the glycocalyx. Solid tumors also have a dynamic fluid microenvironment with elevated interstitial flow. In the present work we further investigate the hypothesis that interstitial flow is sensed by the tumor glycocalyx leading to activation of cell motility and metastasis. Using a highly metastatic renal carcinoma cell line (SN12L1) and its low metastatic counterpart (SN12C) we demonstrate in vitro that the small molecule Suberoylanilide Hydroxamic Acid (SAHA) inhibits the heparan sulfate synthesis enzyme N-deacetylase-N-sulfotransferase-1, reduces heparan sulfate in the glycocalyx and suppresses SN12L1 motility in response to interstitial flow. SN12L1 cells implanted in the kidney capsule of SCID mice formed large primary tumors and metastasized to distant organs, but when treated with SAHA metastases were not detected. In another set of experiments, the role of hyaluronic acid was investigated. Hyaluronan synthase 1, a critical enzyme in the synthetic pathway for hyaluronic acid, was knocked down in SN12L1 cells and in vitro experiments revealed inhibition of interstitial flow induced migration. Subsequently these cells were implanted in mouse kidneys and no distant metastases were detected. These findings suggest new therapeutic approaches to the treatment of kidney carcinoma metastasis.
Project description:Dimethyl sulfoxide (DMSO) is widely used as a solvent for water-insoluble substances and has biological properties such as inducing cellular differentiation, scavenging free radicals, and acting as a radioprotectant. DMSO has also been reported to enhance mineralization in osteoblastic cells. However, its molecular mechanisms have not yet been fully elucidated. In this study, we identified the differentially expressed genes associated with dimethyl sulfoxide-enhanced mineralization in mouse MC3T3-E1 preosteoblast cells using GeneChip® oligonucleotide microarrays.
Project description:Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells with strong immunosuppressive activity that promote tumor growth. In this study, we describe a mechanism by which cancer cells control MDSCs in human cancers by upregulating TRF2, a protein required for telomere stability. Specifically, we showed that the TRF2 upregulation in cancer cells has extratelomeric roles in activating the expression of a network of genes involved in the biosynthesis of heparan sulfate proteoglycan, leading to profound changes in glycocalyx length and stiffness, as revealed by atomic force microscopy. This TRF2-dependent regulation facilitated the recruitment of MDSCs, their activation via the TLR2/MyD88/IL-6/STAT3 pathway leading to the inhibition of natural killer recruitment and cytotoxicity, and ultimately tumor progression and metastasis. The clinical relevance of these findings is supported by our analysis of cancer cohorts, which showed a correlation between high TRF2 expression and MDSC infiltration, which was inversely correlated with overall patient survival.
Project description:Triple SILAC phosphoproteome experiment of hormone deprived MCF7 cells treated with estradiol (E2) in presence or absence of the PLK1 inhibitor BI2536. Conditions were as follows: light (L) population: 1h DMSO, 30min EtOH; medium (M) population: 1h DMSO, 30min E2; heavy (H) population: 1h BI2536, 30min E2. Biological replicates: A and B. Phosphopeptides were enriched from soluble and insoluble fraction using a two step enrichment protocol consiting of strong cation exchange and TiO2 affinity chromatography. The peptides were analyzed on an Orbitrap Velos mass spectrometer linked to an online nanoflow HPLC system via a nanoelectrospray ion source and fragmented via higher-energy C-trap dissociation (HCD). Analysis was carried out using MaxQuant (v. 1.2.2.5) with standard settings including STY phosphporylation as variable modification.
Project description:An important question for the use of the mouse as a model for studying human disease is the degree of functional conservation of genetic control pathways from human to mouse. The human and mouse placenta show structural similarities but there have been no systematic attempt to assess their molecular similarities or differences. We built a comprehensive database of protein and microarray data for the highly vascular exchange region micro-dissected from the human and mouse placenta near-term. Abnormalities in this region are associated with two of the most common and serious complications of human pregnancy, maternal preeclampsia (PE) and fetal intrauterine growth restriction (IUGR), each disorder affecting ~5% of all pregnancies. Over 7,000 orthologs were detected with 70% co-expressed and over 80% of genes known to cause placental phenotypes in mouse were co-expressed. These genes form a tight protein-protein interaction network with novel candidate genes likely to be important in placental structure and/or function. The entire data is available as a web-accessible database to guide the informed development of mouse models to study human disease This experiment is now fully represented in NCBI Peptidome database with accession PSE115; http://www.ncbi.nlm.nih.gov/peptidome/search/index.shtml?acc=PSE115 Microdissection of human villous trees and mouse placental labyrinth. Tissues were split for microarray and protein analysis. For protein analysis samples were first fractionated by differential sucrose gradients into mitochrondria, cytosol, microsomes and nuclei. Mitochrondira and neuclei were each extracted by two different methods for soluble and insoluble material. Each subcellular fraction for each tissue was analysed in quintuplet by 9 step 2 dimensional LC/MSMS. This generated a total of 270 mzXML files for each tissue.
Project description:MS based secretome analyses of Strongyloides venezuelensis, a gastrointestinal parasite of rats that is widely used as a laboratory model and is known to produce both soluble and insoluble (adhesive) secretions during its parasitic stages.
Project description:1. Quantitative Proteomics: MDA-MB-231, MDA-MB-468, and MCF12A cells were treated with DMSO (vehicle control) or SU056 (novel small molecule drug candidate). Quantitative proteomics analysis was performed on cell lysates. 2. Cellular Thermal Shift Assay (CETSA): MDA-MB-231 cells were treated with DMSO or SU056 and incubated at different temperatures and protein differences in the resulting soluble and insoluble fractions were determined.3. Cellular Thermal Shift Assay (CETSA): MDA-MB-231 YBOX1 KD cells were treated with DMSO or SU056 and incubated at different temperatures and protein differences in the soluble fractions were determined.
Project description:The objective of the study is to profile histone H3 lysine nine di-methylation (H3K9me2) in Arabidopsis thaliana and to correlate it with DNA methylation. We constructed a high-resolution genome-wide map of H3K9me2 methylation by using native chromatin immunoprecipitation coupled with HD2 whole genome Nimblegen microarrays. Three replicas were performed for the native ChIP. As a control, one replica of ChIP isolated from crosslinked tissue was used.