Endothelial albumin binding proteins are membrane-associated components exposed on the cell surface

The heterobifunctional, photoactivatable, thiol-cleavable cross-linker sulfosuccinimidyl 2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate (SASD) was radioiodinated and used to determine whether endothelial albumin binding proteins (ABP) recently identified (Ghinea, N., Fixman, A., Alexandru, D., Popov, D., Hasu, M., Ghitescu, L., Eskenasy, M., Simionescu, M., and Simionescu, N. (1988) J. Cell Biol. 107, 231-239) are plasma membrane-associated components exposed on the cell surface. Microvascular endothelial cells (MEC) freshly isolated from rat epididymal fat were incubated with 125I-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (ASD)-albumin conjugate which upon photolysis by UV light was cross-linked to the receptor proteins. By cleaving the disulfide linkages of the cross-linker with 5% beta-mercaptoethanol and the ligand-receptor interactions with 0.1% sodium dodecyl sulfate, the radioiodinated ASD moiety remained attached to the receptor peptides which were further detected by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. In parallel, samples were examined by ligand blotting with albumin-gold complex. The results showed that in these experimental conditions ABP are represented by two major peptides of 31 and 18 kDa and two minor bands of 73 and 56 kDa. Densitometric scanning showed that the two major bands constitute more than 70% of the total ABP. The four peptides were not apparent if the samples were not UV-irradiated. The binding of the radioiodinated ligand to ABPs was reduced by approximately 82% in the presence of excess competitive unlabeled albumin. When MEC were incubated with unlabeled SASD and exposed to UV light, the autoradiographic banding pattern obtained was similar to that of either radioiodinated receptor proteins or MEC not treated with SASD. This indicated that the four albumin binding peptides are distinct proteins of the endothelial cell plasmalemma.     

EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids

The four Eps15 homology (EH) domain-containing proteins, EHD1-EHD4, have recently been ascribed roles in the regulation of the recycling of distinct receptor molecules and are often found associated with tubular structures. Here, we report the analysis of all four EHD proteins with regard to tissue distribution, intracellular localization and lipid binding properties. Specific antibodies reveal distinct expression profiles for the individual proteins in tissues and at intracellular locations, where they potentially interact with specific phospholipids. Moreover, EHD proteins colocalize with vesicular and tubular structures, implying roles in transport processes and cytoskeletal dynamics. Protein variants carrying mutations in the N-terminal nucleotide-binding P-loop region are no longer associated with phospholipids or membrane compartments, while deletion of the C-terminal EH domain affects targeting to tubular structures. All EHD proteins are able to bind to phospholipids, but localizations differ for each protein.     

Wnt family proteins are secreted and associated with the cell surface.

Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1.     

Induction of stress proteins in response to hypoxia

Hypoxia is a severe stress factor to which man and most other mammalian species are capable of adapting. However, the cellular mechanism which enable cells to adapt are still unknown. Effect of hypoxia was studied on the synthesis of hypoxia induced proteins in rat kidney and in vero cell line (monkey kidney). These were exposed to hypoxia at 240 mmHg pressure for 1 hr. The induction of stress protein was determined by probing with monoclonal antibodies against 65 kDa heat shock protein (hsp65). The induction of a 65 kDa protein was 3.6 fold higher to the total cellular protein, both in cell lines and kidney of rats. In vivo response was predominantly observed in renal cortical region particularly in glomeruli. The induction of stress proteins during hypoxia suggests their importance in the maintenance of cellular integrity under hypoxia.     

Endothelial cell metabolism in health and disease: impact of hypoxia

In contrast to the general belief, endothelial cell (EC) metabolism has recently been identified as a driver rather than a bystander effect of angiogenesis in health and disease. Indeed, different EC subtypes present with distinct metabolic properties, which determine their function in angiogenesis upon growth factor stimulation. One of the main stimulators of angiogenesis is hypoxia, frequently observed in disease settings such as cancer and atherosclerosis. It has long been established that hypoxic signalling and metabolism changes are highly interlinked. In this review, we will provide an overview of the literature and recent findings on hypoxia‐driven EC function and metabolism in health and disease. We summarize evidence on metabolic crosstalk between different hypoxic cell types with ECs and suggest new metabolic targets.     

Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.     

Arabidopsis WPP-domain proteins are developmentally associated with the nuclear envelope and promote cell division

The nuclear envelope (NE) acts as a selective barrier to macromolecule trafficking between the nucleus and the cytoplasm and undergoes a complex reorganization during mitosis. Different eukaryotic kingdoms show specializations in NE function and composition. In contrast with vertebrates, the protein composition of the NE and the function of NE proteins are barely understood in plants. MFP1 attachment factor 1 (MAF1) is a plant-specific NE-associated protein first identified in tomato (Lycopersicon esculentum). Here, we demonstrate that two Arabidopsis thaliana MAF1 homologs, WPP1 and WPP2, are associated with the NE specifically in undifferentiated cells of the root tip. Reentry into cell cycle after callus induction from differentiated root segments reprograms their NE association. Based on green fluorescent protein fusions and immunogold labeling data, the proteins are associated with the outer NE and the nuclear pores in interphase cells and with the immature cell plate during cytokinesis. RNA interference-based suppression of the Arabidopsis WPP family causes shorter primary roots, a reduced number of lateral roots, and reduced mitotic activity of the root meristem. Together, these data demonstrate the existence of regulated NE targeting in plants and identify a class of plant-specific NE proteins involved in mitotic activity.     

Endothelial cell phagokinesis in response to specific metal ions

Salts of Cu^{I, II}, Ni^II, Sn^II, In^III and a sub-fraction of the cupoprotein ceruloplasmin induced phagokinesis of cultured aortal endothelial cells. A variant aortal endothelial cell line was highly sensitive; cells travelled up to 1000 μm in 24 h in response to 2× 10^?6 M SnCl₂. Other metal ions tested (Zn^II, Co^II, Mn^II, Cr^II, Fe^III, Al^III, Sb^III and Mo^II) were not active. The motility response of endothelial cells to Cu ions in vitro is proposed as a model system for studying early events in neovascularization and as a sensitive assay for detecting angiogenic activity in fractions from cells and tissues.     

Endothelial cell chemotaxic activity expressed in rat placenta is not associated with prolactin-like proteins B and C.

Conditioned medium from gestation day 18 rat placental cultures showed potent stimulation of the directional migration of human retinal endothelial cells. To examine the role of major secreted placental proteins in this chemotaxic activity, prolactin-like proteins (PLPs)-B and C were purified from rat placenta using immuno-affinity chromatography. In contrast to conditioned medium, native PLP-B and PLP-C preparations failed to show any significant stimulation of endothelial cell migration. This study further examined the ability of PLP-B to bind to rat receptors for growth hormone (GH-R) and prolactin (PRL-R). In competitive binding assays with [125I]-hGH, neither native nor recombinant PLP-B preparations showed significant high affinity binding to the transfected rat GH-R or PRL-R. In summary, neither PLP-B nor PLP-C exhibit the potent chemotaxis stimulatory activity of placental conditioned media, nor does PLP-B show evidence of ability to act via rat GH or PRL receptors.     

Tyrosinase family proteins are antigens specific to Vogt-Koyanagi-Harada disease

Vogt-Koyanagi-Harada (VKH) disease (and sympathetic ophthalmia) is an ocular inflammatory disease that is considered to be a cell-mediated autoimmune disease against melanocytes. The purpose of this study was to determine the Ags specific to VKH disease and to develop an animal model of VKH disease. We found that exposure of lymphocytes from patients with VKH disease to peptides (30-mer) derived from the tyrosinase family proteins led to significant proliferation of the lymphocytes. Immunization of these peptides into pigmented rats induced ocular and extraocular changes that highly resembled human VKH disease, and we suggest that an experimental VKH disease was induced in these rats. We conclude that VKH disease is an autoimmune disease against the tyrosinase family proteins.     

Tissue specific expression of cell wall proteins of seedlings of Prosopis chilensis during development and wound stress

Four cell wall proteins of seedling cotyledons of Prosopis chilensis were characterized by SDS-polyacrylamide gel electrophoresis. The molecular masses of these proteins were 180, 126, 107 and 63 kDa. All of them immuno-crossreacted with polyclonal antibodies raised against extensin from soybean seed coats. Immuno-dot blot analysis demonstrated that the minimum expression of cotyledonary cell wall proteins was 48 h after seed imbibition, while 24 h after wound stress the expression of these wall proteins increased four-fold. Tissue immuno-prints and immuno-histochemistry showed that the proteins are expressed in the cell wall of all tissues. However, the epidermis and vascular bundles of cotyledons, hypocotyls and roots, and the living cells surrounding the wounded areas highly expressed the wall proteins. When the primary roots of the seedlings were injured by performing cuts with razor blades, the seedlings achieved a growth three times faster than control seedlings and secondary roots developed in sites close to the injuries. Immuno-histochemistry of secondary roots revealed that the root tips and the area of the cortical tissue of the primary roots being pressed by the the emerging root tip, highly expressed the cell wall proteins.     

Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation

Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM (35)S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.     

Endothelial cell fatty acid unsaturation mediates cold-induced oxidative stress

Ultraprofound hypothermia (< 5 degrees C) induces changes to cell membranes such as liquid-to-gel lipid transitions and oxidative stress that have a negative effect on membrane function and cell survival. We hypothesized that fatty acid substitution of endothelial cell lipids and alterations in their unsaturation would modify cell survival at 0 degrees C, a temperature commonly used during storage and transportation of isolated cells or tissues and organs used in transplantation. Confluent bovine aortic endothelial cells were treated with 18-carbon fatty acids (C18:0, C18:1n-9, C18:2n-6, or C18:3n-3), C20:5n-3 or C22:6n-3 (DHA), and then stored at 0 degrees C without fatty acid supplements. Storage of control cells caused the release of lactate dehydrogenase (LDH) and a threefold increase in lipid peroxidation (LPO) when compared to control cells not exposed to cold. Pre-treating cells with C18:0 decreased the unsaturation of cell lipids and reduced LDH release at 0 degrees C by 50%, but all mono- or poly-unsaturated fatty acids increased injury in a concentration-dependent manner and as the extent of fatty acid unsaturation increased. DHA-treatment increased cell fatty acid unsaturation and caused maximal injury at 0 degrees C, which was prevented by lipophilic antioxidants BHT or vitamin E, the iron chelator deferoxamine, and to a lesser extent by vitamin C. Furthermore, the cold-induced increase in LPO was reduced by C18:0, vitamin E, or DFO but enhanced by DHA. In conclusion, the findings implicate iron catalyzed free radicals and LPO as a predominant mechanism of endothelial cell injury at 0 degrees C, which may be reduced by increasing lipid saturation or treating cells with antioxidants.     

Smooth muscle adherens junctions associated proteins are stable at the cell periphery during relaxation and activation

This study was performed to determine the stability of the adherens junction (AJ)-associated proteins at the smooth muscle cell (SMC) plasma membrane during relaxing and activating conditions. Dog stomach, ileum, colon, and trachea tissues were stored in Ca²⁺-free PSS or regular PSS or were activated in 10 µM carbachol in PSS before rapid freezing. The tissues were subsequently sectioned and immunoreacted using antibodies for vinculin, talin, fibronectin, and caveolin to determine their cellular distribution in these tissues under these conditions. In all four tissues and under all three conditions, the distribution of these four proteins remained localized to the periphery of the cell. In transverse tissue sections, the AJ-associated proteins formed a distinct punctate pattern around the periphery of the SMCs at the plasma membrane. These domains alternated with the caveolae (as identified by the presence of caveolin). In longitudinal tissue sections, the AJ-associated proteins formed continuous tracks or staves, while the caveolae remained punctate in this dimension as well. Caveolin is not present in the tapered ends of the SMCs, where the AJ-associated proteins appear continuous around the periphery. Densitometry of the fluorophore distribution of these proteins showed no shift in their localization from the SMC periphery when the tissues were relaxed or when they were activated before freezing. These results suggest that under physiologically relaxing and activating conditions, AJ-associated proteins remain stably localized at the plasma membrane. vinculin; talin; fibronectin; caveolin; stomach; ileum; colon; trachea