Proteomic exploitation on prothymosin alpha-induced mononuclear cell activation

Prothymosin alpha (ProTalpha) is an acidic polypeptide associated both with cell proliferation and immune regulation. Although ProTalpha's immunomodulating activity is well established at cellular level, limited information is available regarding the signaling pathways triggered by ProTalpha. Using 2-DE proteomic technology, we investigated changes in protein expression of ProTalpha-stimulated peripheral blood mononuclear cells (PBMC) in the course of a 3-day incubation. Using healthy donor- and cancer patient-derived PBMC, 12 gels were studied, identifying 53 differing protein spots via PMF comparison analysis. Among others, we identified interleukin-1 receptor-associated kinase 4, heat-shock protein 90, lipocalin 2, ribophorin 1, eukaryotic elongation factor 2, 14-3-3 protein, L-plastin, and MX2 protein, all of which were found to be overexpressed upon ProTalpha activation. Based on the physiological role of upregulated proteins, we propose the following model for ProTalpha's immunological mode of action: on day 1, ProTalpha triggers monocyte activation, possibly via toll-like receptor signaling, and enhances antigen presentation, consequently promoting and stabilizing monocyte-T-cell immune synapse; on day 2, activated monocytes produce interleukin (IL)-1, while T-cell receptor triggering promotes T-cell proliferation and IL-2 production; finally, on day 3, ProTalpha-activated PBMC express proteins related to adhesion and cytotoxic effector functions, both contributing to the increase of their lytic activity.     

Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.     

Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells

We used affinity chromatography to isolate a specific laminin-binding protein from murine fibrosarcoma cells. These cells bind exogenous laminin to their surface with high affinity (Kd = 2 X 10(-9)M for laminin) with approximately 5 X 10(4) sites per cell. Laminin affinity chromatography of [35S]methionine-labeled cell extracts produced two distinct proteins. One was identified as Type IV (basement membrane) collagen based on its migration pattern on SDS gels and bacterial collagenase sensitivity. The other protein, which migrates as a single band or closely spaced doublet on reduced SDS gels, has a reduced molecular weight of 69,000. Using a nitrocellulose filter disk assay, we found that the latter protein specifically bound 125I-laminin with the same high affinity (Kd = 2 X 10(-9)M for laminin) as did intact fibrosarcoma cells. By iodinating intact cells, we demonstrated that this laminin-binding protein is on the cell surface. We conclude that this protein with reduced molecular weight of 69,000 is a subunit or component of a larger cell surface receptor protein for laminin in this fibrosarcoma model. This laminin receptor may mediate the interaction of the cell with its extracellular matrix.     

Prothymosin alpha modulates the interaction of histone H1 with chromatin.

Prothymosin alpha (ProTalpha) is an abundant acidic nuclear protein that may be involved in cell proliferation. In our search for its cellular partners, we have recently found that ProTalpha binds to linker histone H1. We now provide further evidence for the physiological relevance of this interaction by immunoisolation of a histone H1-ProTalpha complex from NIH 3T3 cell extracts. A detailed analysis of the interaction between the two proteins suggests contacts between the acidic region of ProTalpha and histone H1. In the context of a physiological chromatin reconstitution reaction, the presence of ProTalpha does not affect incorporation of an amount of histone H1 sufficient to increase the nucleosome repeat length by 20 bp, but prevents association of all further H1. Consistent with this finding, a fraction of histone H1 is released when H1-containing chromatin is challenged with ProTalpha. These results imply at least two different interaction modes of H1 with chromatin, which can be distinguished by their sensitivity to ProTalpha. The properties of ProTalpha suggest a role in fine tuning the stoichiometry and/or mode of interaction of H1 with chromatin.     

The search for the chaperonin 60 receptors

Chaperonin (Cpn)60 proteins have the ability to activate human and murine myeloid cells. There is contradictory evidence that the receptor for this protein is either similar to that of lipopolysaccharide--CD14 and one or other toll-like receptor (e.g. TLR4) or is some other, undidentified, receptor. In an attempt to directly identify the receptor for Mycobacterium tuberculosis Cpn60.1 we have used two approaches. The first is to use Cpn60.1 as an affinity ligand to pull out the receptor from lysates of the murine monocyte cell line RAW 264.7. The second is to crosslink Cpn60.1 to its receptor on RAW cells and isolate the complex by immunoprecipitation. These methods have worked for other receptors. Using affinity chromatography, 2D SDS-PAGE and peptide mass fingerprinting with MALDI-TOF MS it was found that a number of proteins had the ability to bind to Cpn60.1 on an affinity matrix. We identified five proteins, three of which were likely to be on the cell surface. One of these proteins, the endoplasmic reticulum molecular chaperone, BiP did bind to Cpn60.1 with low affinity. Protein crosslinking studies proved inadequate as insufficient protein could be isolated for mass spectrometric identification. Thus, it appears that Cpn60.1, like Hsp70, may bind to a number of cell surface proteins. BiP appears to be one of these receptor proteins but more work is needed to identify those responsible for signalling. Of interest, CD14 and TLR4 were not identified in this study as a receptor for Cpn60.1.     

A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.     

Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fc gamma RI).

The high affinity receptor that binds the Fc domain of immunoglobulin G (IgG) subclasses 1 and 3 (Fc gamma RI) mediates important immune defense functions by inducing cell surface changes on human leukocytes. In this article, we document direct high affinity binding of Fc gamma RI to the actin filament cross-linking protein, actin-binding protein (ABP). In the absence of IgG, all Fc gamma RI molecules in undifferentiated cells of myeloid line U937 bound to ABP over a 9-fold range of Fc gamma RI expression induced by human IFN-gamma. Binding of IgG to U937 cells constitutively expressing Fc gamma RI or to COS cells genetically transfected to express Fc gamma RI rapidly decreased the avidity of Fc gamma RI for ABP. This finding suggests the existence of a pathway communicating a signal between a functional IgG receptor and intracellular components involved in the effector responses to Fc gamma RI-ligand interaction.     

Amiloride inhibits constitutive internalization and increases the surface number of epidermal growth factor receptors in intact rat hepatocytes

In previous experiments the surface expression of epidermal growth factor (EGF) receptors in freshly isolated rat hepatocytes varied temperature- and time-dependently and was depleted by monensin and cycloheximide in a way suggesting that a subpopulation of these receptors are subject to constitutive cycling (Gladhaug and Christoffersen; 1988). We here report the finding that pretreatment of the hepatocytes with amiloride exerts marked effects on cellular EGF receptor movements. After 2 h incubation with 1 mM amiloride, the receptor level was approximately 270,000 sites/cell surface vs. 140,000 in the untreated cell, with no change in receptor affinity. Amiloride thus stabilized the surface EGF receptor pool at an elevated level. In cells pretreated with amiloride for 60 min, the relative endocytosis decreased from about 2.6 EGF molecules internalized per receptor during 15 min endocytosis in untreated cells to about 1.5 molecules/receptor in amiloride-treated cells. These results suggest that amiloride causes an accumulation of EGF receptors at the hepatocyte surface due to inhibition of constitutive receptor internalization. In addition, it was found that in amiloride-treated hepatocytes the phorbol ester TPA strongly inhibited high-affinity EGF binding without affecting the total surface receptor number. In control cells, TPA did not consistently affect binding. Pretreatment with amiloride prevented surface EGF receptor depletion induced by cycloheximide and puromycin, but it did not significantly inhibit surface receptor depletion caused by monensin. Although the underlying mechanism of the amiloride effect on intracellular receptor trafficking is not clear, the results provide further evidence for a continuous, ligand-independent EGF receptor cycling pathway in hepatocytes.     

Identification of prothymosin-alpha1, the necrosis-apoptosis switch molecule in cortical neuronal cultures

We initially identified a nuclear protein, prothymosin-alpha1 (ProTalpha), as a key protein inhibiting necrosis by subjecting conditioned media from serum-free cultures of cortical neurons to a few chromatography steps. ProTalpha inhibited necrosis of cultured neurons by preventing rapid loss of cellular adenosine triphosphate levels by reversing the decreased membrane localization of glucose transporters but caused apoptosis through up-regulation of proapoptotic Bcl(2)-family proteins. The apoptosis caused by ProTalpha was further inhibited by growth factors, including brain-derived neurotrophic factor. The ProTalpha-induced cell death mode switch from necrosis to apoptosis was also reproduced in experimental ischemia-reperfusion culture experiments, although the apoptosis level was markedly reduced, possibly because of the presence of growth factors in the reperfused serum. Knock down of PKCbeta(II) expression prevented this cell death mode switch. Collectively, these results suggest that ProTalpha is an extracellular signal protein that acts as a cell death mode switch and could be a promising candidate for preventing brain strokes with the help of known apoptosis inhibitors.     

Endocytosis of N-acetylglucosamine-containing glycoproteins by rat fibroblasts expressing a single species of chicken liver glycoprotein receptor

A cDNA clone for the chicken liver receptor which mediates endocytosis of glycoproteins containing terminal N-acetylglucosamine has been isolated and sequenced, confirming the previously obtained amino acid sequence of this protein (which is also known as the chicken hepatic lectin). This cDNA was introduced into Rat-1 fibroblasts and expressed using the promotor in the long terminal repeat of Moloney murine leukemia virus. Cells expressing chicken receptor were identified by screening with antireceptor antibodies followed by fluorescein-conjugated second antibodies. Receptor expressed in these cells was indistinguishable on gel electrophoresis from receptor isolated from liver. Three clonally isolated lines were examined for their ability to bind agalacto-alpha 1-acid glycoproteins at 0 degrees C and to take up and degrade this ligand at 37 degrees C. The receptor number (50,000/cell), affinity for ligand (35 nM), and uptake rate (5 molecules ligand/surface receptor/h) are similar to those previously observed for chicken hepatocytes, and for the uptake of asialoglycoproteins by rat hepatocytes and hepatoma cells. These findings indicate that the chicken receptor correctly traverses the endocytic pathway in a rat cell even though the cytoplasmic domain of this protein shows no primary structural homology with the corresponding portion of the rat liver receptor or with receptors found in fibroblasts.