scholarly journals Most iodinatable fibroblast surface proteins accompany the cytoplast membrane during cytochalasin B-mediated enucleation of chick embryo fibroblasts.

1982 ◽  
Vol 94 (3) ◽  
pp. 557-564 ◽  
Author(s):  
D P Witt ◽  
J A Gordon
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Preliminary Evidence ◽  
Surface Proteins ◽  
Cell Monolayers ◽  
Low Salt ◽  
Embryo Fibroblasts ◽  
Labeling Pattern ◽  
Chicken Embryo Fibroblasts ◽  
Serum Components

Six different proteins are found to be reproducibly exposed on the cell surface of chicken embryo fibroblasts (CEF) by the criterion of lactoperoxidase-catalyzed iodination (250,000, 185,000, 130,000, 100,000, 87,000, and 75,000 daltons). We wondered whether cell enucleation might lead to a differential partition of these surface proteins with the karyoplast or cytoplast membrane. We found that there is a marked enrichment of most iodinatable cell surface proteins in the cytoplast after cytochalasin-mediated enucleation of cell monolayers. Nearly all the iodinatable fibronectin remains with the cytoplast. Of the six labeled proteins, the karyoplast membrane contains a small amount of the 130 kdalton protein as well as trace levels of the 100-, 85-, and 75-kdalton proteins. Proteolysis or selective shedding of membrane proteins were not significant factors in the relative exclusion of iodinatable membrane proteins from the karyoplast. The cytoplast could replace some exposed membrane proteins after removal by trypsinization; however, fibronectin was not detectable within 10 h. That the karyoplast was not capable of membrane protein synthesis and/or insertion was suggested by the lack of any change in the labeling pattern of karyoplasts up to 8-h incubation after enucleation. A variety of control studies indicated that the surface proteins identified in this report were cell-derived and not adsorbed serum components. That some of the iodinatable proteins are intrinsic membrane proteins was suggested by their resistance to removal by conditions thought to extract extrinsic membrane proteins (i.e., low salt, high salt, and NaOH washes). lack of effect of cytoskeletal disrupting agents (preliminary evidence) suggests the nonrandom partition of membrane proteins may depend on anchoring of membrane proteins by a system(s) in the cytoplast other than intact microtubules and microfilaments.

scholarly journals Labile disulfide bonds are common at the leucocyte cell surface

Open Biology ◽  
2011 ◽  
Vol 1 (3) ◽  
pp. 110010 ◽  
Author(s):  
Clive Metcalfe ◽  
Peter Cresswell ◽  
Laura Ciaccia ◽  
Benjamin Thomas ◽  
A. Neil Barclay
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Disulfide Bonds ◽  
Reduction Process ◽  
Cysteine Residue ◽  
Surface Proteins ◽  
Protein Activity ◽  
Functional Changes ◽  
Reducing Conditions

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism.

scholarly journals Salmonella typhimurium induces selective aggregation and internalization of host cell surface proteins during invasion of epithelial cells

1994 ◽  
Vol 107 (7) ◽  
pp. 2005-2020 ◽  
Author(s):  
F. Garcia-del Portillo ◽  
M.G. Pucciarelli ◽  
W.A. Jefferies ◽  
B.B. Finlay
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Heavy Chain ◽  
Surface Proteins ◽  
Class I ◽  
Plasma Membrane Proteins ◽  
Class I Mhc ◽  
Host Plasma Membrane

Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, beta 2-microglobulin, fibronectin-receptor (alpha 5 beta 1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to beta 1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia-internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.

scholarly journals Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines

2019 ◽  
Author(s):  
Bernadette Jeremiasse ◽  
Csaba Matta ◽  
Christopher R. Fellows ◽  
David J. Boocock ◽  
Julia R. Smith ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Inflammatory Cytokines ◽  
Ionic Composition ◽  
Interleukin 1 ◽  
Surface Proteins ◽  
Low Grade ◽  
Necrosis Factor Alpha ◽  
Cell Surface Proteins ◽  
Pro Inflammatory Cytokines

Abstract Background: Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the ‘surfaceome’. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). Methods: We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Results: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different levels of expression following chondrocyte stimulation with pro-inflammatory cytokines.LPR-1, thrombospondin, VDAC1-2 and annexin A1 were considered to be of special interest and were analysed further and validated by western blotting. Conclusions: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.

scholarly journals Characterization of a 140-kD avian cell surface antigen as a fibronectin-binding molecule.

1986 ◽  
Vol 102 (2) ◽  
pp. 442-448 ◽  
Author(s):  
S K Akiyama ◽  
S S Yamada ◽  
K M Yamada
Keyword(s):  
Cell Surface ◽  
Surface Antigen ◽  
Chicken Embryo ◽  
Signal Sequence ◽  
Cell Surface Antigen ◽  
Affinity Column ◽  
Fibronectin Receptor ◽  
Experimental Systems ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

A 140,000-D protein cell surface antigen (140k) complex has been implicated in fibronectin-mediated cell-substratum attachment. We have used three different experimental systems to evaluate the hypothesis that this 140k complex can function as a fibronectin receptor. A monoclonal antibody that binds to the 140k complex specifically inhibits the direct binding of 3H-labeled 75,000-D fibronectin cell-binding fragment (f75k) to chicken embryo fibroblasts in suspension. The 140k complex is retarded in its passage through an affinity column consisting of immobilized f75k, and this interaction is specifically inhibited by a synthetic peptide that contains the fibronectin cell-recognition signal sequence. Finally, exogenous purified 140k complex inhibits the attachment and spreading of chicken embryo fibroblasts on fibronectin-coated substrates. Thus, our results indicate that the 140k complex can bind directly to fibronectin and is likely to be a fibronectin receptor for chicken cells.

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