Global analysis of protein phosphorylation networks in insulin signaling by sequential enrichment of phosphoproteins and phosphopeptides.

Although the important role of protein phosphorylation in insulin signaling networks is well recognized, its analysis in vivo has not been pursued in a systematic fashion through proteome-wide studies. Here we undertake a global analysis of insulin-induced changes in the rat liver cytoplasmic and endosomal phosphoproteome by sequential enrichment of phosphoproteins and phosphopeptides. After subcellular fractionation proteins were denatured and loaded onto iminodiacetic acid-modified Sepharose with immobilized Al³⁺ ions (IMAC-Al resin).

Erythropoietin-dependent endothelial proteins: potential use against erythropoietin resistance.

The endothelium was the first non-hematopoietic tissue to be identified as a physiological target for erythropoietin (EPO). EPO is involved in recruitment and mobilization of endothelial progenitors and stimulates the production of erythroid cell regulatory factors in endothelial cells. Production of these EPO-dependent factors is inhibited by IL-3 in vitro.

Biological activities and molecular interactions of the C-terminal residue of thrombospondin-4, an epitome of acidic amphipathic peptides.

C21, the C-terminal residue of thrombospondin-4 (TSP-4), was identified as a peptide growth factor during an investigation concerning erythropoietin-dependent, erythroid stimulating factors of endothelial origin. It is active in cultures of several human hematopoietic stem cells, skin fibroblasts and kidney epithelial cells and stimulates red cell formation in anemic mice. A method of affinity chromatography in the presence of high concentrations of Triton X-100, previously developed for identifying proteins associated with the TSP-1 receptor CD47, was utilized for the detection of C21 binding molecules and their detergent-resistant, associated partners.

Quantitative proteomics of nasal mucus in chronic sinusitis with nasal polyposis.

Background: Proteomics has been used as a tool for identification of the protein content of nasal mucus in diseased and healthy subjects. Thirty-five proteins in both chronic rhinosinusitis (CRS) and control groups were identified in a previous study by our group using conventional mass spectrometry analysis. Ten of these proteins were related to innate and acquired immunity and showed differences in expression between the two groups.

Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin.

Proteomics of nasal mucus in chronic rhinosinusitis.

Background: Chronic rhinosinusitis (CRS) is among the three most common chronic diseases in North America. The area of proteomics research is providing tremendous insight into the mechanisms of a variety of physiological processes and disease states. The purpose of this study was to evaluate qualitative and quantitative differences in protein content of nasal mucus in patients with chronic hypertrophic sinusitis with nasal polyposis when compared with control subjects.

Thrombospondin 1, produced by endothelial cells under the action of erythropoietin, stimulates thymidine incorporation into erythroid cells and counteracts the inhibitory action of insulin-like growth factor binding protein 3.

The nature of erythropoietin (EPO)-dependent, erythroid cell regulatory factors secreted by endothelial cells is largely unknown. The production of thrombospondin 1 (TSP-1) and insulin-like growth factor binding protein 3 (IGFBP-3) is increased in cultures of human umbilical vein endothelial cells (HUVEC) incubated with erythropoietin (EPO). Simultaneous incubation of HUVEC with EPO and interleukin 3 (IL-3) resulted in a decreased production, suggesting that both TSP-1 and IGFBP-3 belong to the EPO- and IL-3-dependent erythroid regulatory factors previously described in cultures of bone marrow endothelial cells.

The C-terminal peptide of thrombospondin-4 stimulates erythroid cell proliferation.

Erythropoietin (EPO) stimulates the production of small erythroid cell stimulating factors (molecular weight <5 kDa) in cultures of bone marrow endothelial cells. We identified a fragment of thrombospondin-4 (TSP-4) as an EPO-stimulated protein in endothelial cell lysates. Pre-incubation of the low molecular weight fractions from supernatants of EPO-treated umbilical cord endothelial cells (HUVEC) with antibodies against the C-terminal residues of TSP-1,2 and TSP-4 decreased the erythroid cell stimulating activity.

The fusion of IGF I with stromal cell-derived factor I or alpha1 proteinase inhibitor alters their mitogenic or chemotactic activities while keeping their ability to inhibit HIV-1-gp120 binding.

It has been previously reported that insulin-like growth factor I (IGF I) decreases in AIDS patients with wasting, a condition that is partially prevented by combined IGF I growth hormone therapy. By generating bifunctional proteins of IGF I and stromal cell-derived factor 1alpha (SDF-1alpha) or alpha1 proteinase inhibitor (API), two proteins known to prevent HIV infection, it may be possible to improve the therapeutic effectiveness of these compounds for the treatment of AIDS-mediated wasting. SDF-1alpha or the M351E-M358L mutant of API were attached at the C-terminal end of IGF I and synthesized by a stable insect cell expression technique.

The improved survival of hematopoietic cells cultured with a fusion protein of insulin-like growth factor II (IGF-II) and interleukin 3 (IL-3) is associated with increases in Bcl-xL and phosphatidylinositol-3 kinase activity.

We compared the antiapoptotic activity of a recombinant chimera of insulin-like growth factor II (IGF-II) and interleukin (IL)-3 with the corresponding equimolar mixture of the individual components based on changes in several factors associated with survival in the CD34+ human hematopoietic cell line TF-1. Propidium iodide-stained cells analyzed by fluorescein-activated cell sorter indicated that the chimera was more effective than the corresponding equimolar mixture in decreasing the amounts of apoptotic cells and increasing the proportion of cells in the S-phase of the cell cycle. The chimera was more effective in increasing the antiapoptotic protein Bclx(L) and produced a significant increase in signal transducer and activator of transcription-5 phosphorylation and in phosphatidylinositol-3 kinase (PI-3K) activity.

Potentiation of hematopoietic cell migration with an IGF-interleukin-3 fusion protein.

A chimera of an N-terminally modified insulin growth factor (IGF)-II, NQPQMVHTY-hIGF-II(9-67) (BOMIGF), fused to interleukin-3 (IL-3) significantly improved the migration of CD34(+) human hematopoietic cells with respect to the effects observed during co-stimulation with BOMIGF and IL-3. A phosphatidylinositol-3 (PI-3) kinase inhibitor specifically inhibited migration in the presence of the chimera, while no significant difference in the inhibition of migration was observed in the presence of a Rho kinase inhibitor. These results suggest a key role of the PI-3 kinase pathway in the potentiation of migration caused by the linkage of BOMIGF and IL-3.

Antagonism between interleukin 3 and erythropoietin in mice with azidothymidine-induced anemia and in bone marrow endothelial cells.

Azidothymidine (AZT)-induced anemia in mice can be reversed by the administration of IGF-IL-3 (fusion protein of insulin-like growth factor II (IGF II) and interleukin 3). Although interleukin 3 (IL-3) and erythropoietin (EPO) are known to act synergistically on hematopoietic cell proliferation in vitro, injection of IGF-IL-3 and EPO in AZT-treated mice resulted in a reduction of red cells and an increase of plasma EPO levels as compared to animals treated with IGF-IL-3 or EPO alone. We tested the hypothesis that the antagonistic effect of IL-3 and EPO on erythroid cells may be mediated by endothelial cells.

Insect cell production of a secreted form of human alpha(1)-proteinase inhibitor as a bifunctional protein which inhibits neutrophil elastase and has growth factor-like activities.

alpha(1)-proteinase inhibitor (API) is a potential therapeutic agent in all diseases in which elastase released by neutrophils has to be effectively neutralized. We ligated the cDNA of human API to the C-terminal section of an insulin-like growth factor II analogue (BOMIGF), known to be properly folded and secreted in insect cells using the baculovirus expression system. The BOMIGF-API chimera was recovered from the incubation medium of the infected cells.