ICEBERG: a novel inhibitor of interleukin-1beta generation.

ProIL-1beta is a proinflammatory cytokine that is proteolytically processed to its active form by caspase-1. Upon receipt of a proinflammatory stimulus, an upstream adaptor, RIP2, binds and oligomerizes caspase-1 zymogen, promoting its autoactivation. ICEBERG is a novel protein that inhibits generation of IL-1beta by interacting with caspase-1 and preventing its association with RIP2. ICEBERG is induced by proinflammatory stimuli, suggesting that it may be part of a negative feedback loop. Consistent with this, enforced retroviral expression of ICEBERG inhibits lipopolysaccharide-induced IL-1beta generation. The structure of ICEBERG reveals it to be a member of the death-domain-fold superfamily. The distribution of surface charge is complementary to the homologous prodomain of caspase-1, suggesting that charge-charge interactions mediate binding of ICEBERG to the prodomain of caspase-1.

Novel peptides selected to bind vascular endothelial growth factor target the receptor-binding site.

Peptides that inhibit binding of vascular endothelial growth factor (VEGF) to its receptors, KDR and Flt-1, have been produced using phage display. Libraries of short disulfide-constrained peptides yielded three distinct classes of peptides that bind to the receptor-binding domain of VEGF with micromolar affinities. The highest affinity peptide was also shown to antagonize VEGF-induced proliferation of primary human umbilical vascular endothelial cells. The peptides bind to a region of VEGF known to contain the contact surface for Flt-1 and the functional determinants for KDR binding. This suggests that the receptor-binding region of VEGF is a binding "hot spot" that is readily targeted by selected peptides and supports earlier assertions that phage-derived peptides frequently target protein-protein interaction sites. Such peptides may lead to the development of pharmacologically useful VEGF antagonists.

Calpromotin, a cytoplasmic protein, is associated with the formation of dense cells in sickle cell anemia.

We have tested the hypothesis that dense cell formation in sickle cell disease is associated with increased binding of calpromotin to the membrane, an event that occurs during the activation of calcium-dependent potassium transport. By SDS polyacrylamide gel electrophoresis, we found that sickle cell membranes contained more calpromotin than did normal membranes when stained with Coomassie brilliant blue or when transferred to nitrocellulose paper and immunostained with horseradish peroxidase. Also, the membranes from dense sickle cells contained significantly (P = 0.00055) higher levels of calpromotin, 2.62+/-1.59 microg/mg membrane protein, compared to light sickle cells, 1.40+/-0.70 microg/mg membrane protein, when measured by an enzyme-linked immunosorbent assay. The ratio of calpromotin associated with dense cell membranes to light cell membranes was significantly greater than 1.0 (P < 0.00005). Transmission electron micrographs of immunogold-labelled membranes supported the increase in calpromotin binding in dense sickle cell membranes. In addition, the immunogold probe demonstrated clustering, which was not observed in light sickle cell membranes nor in normal membranes. Finally, we incubated HbSS cells in vitro using a repetitive deoxygenation/ reoxygenation procedure to produce dense cells and then measured the levels of calpromotin associated with their membranes. As expected, the levels of calpromotin bound to the membrane doubled during the procedure relative to the basal levels at the beginning of the incubation. The correlation coefficient, calculated between the increase in dense cell formation and the increase in calpromotin associated with the membrane, was statistically significant (P = 0.038). The results demonstrate that an increase in calpromotin binding to the membrane is associated with dense cell formation presumably through the activation of the calcium-dependent potassium channel.

Protein 7.2b of human erythrocyte membranes binds to calpromotin.

Calpromotin is a soluble cytoplasmic protein of human red blood cells which is involved in the activation of the charybdotoxin-sensitive calcium-dependent potassium channel. This activation is associated with increased binding of calpromotin to the red cell membrane. To elucidate this mechanism we tested different fractions of red cell membrane proteins to bind to a calpromotin affinity column. Proteins, which bound specifically to the column, were eluted and identified by SDS polyacrylamide gel electrophoresis. This procedure demonstrated that spectrin and actin, from a low salt extraction of the membrane, bound weakly to the column and a portion of this could be attributed to non-specific binding. Glyceraldehyde-3-phosphate dehydrogenase (band 6) and a 40K molecular weight band, from a high salt extraction of the membrane, bound strongly to the affinity column. Several minor integral membrane proteins, obtained by Triton X-100 treatment of the membrane, bound specifically to the calpromotin affinity column. The molecular weight of these proteins ranged from 95k to 23K. We further demonstrated that the 31.5K band from this fraction is protein 7.2b (stomatin) by staining with a monoclonal antibody. Protein 7.2b is believed to have a role in regulating monovalent cation transport through the erythrocyte membrane.

Reconstitution of Ca(2+)-dependent K+ transport in erythrocyte membrane vesicles requires a cytoplasmic protein.

We have demonstrated that calcium-dependent potassium transport in erythrocytes requires the participation of a cytoplasmic protein. Activation of calcium-dependent potassium transport causes an increase in the membrane-bound levels of this protein which is dependent on the calcium concentration and which is highly correlated (r = 0.791, p less than 0.0001) with the loss of potassium. Reconstitution of this transport pathway in sonicated erythrocyte membrane vesicles was achieved only in vesicles containing the cytoplasmic protein indicating a causal relationship in this transport system. The protein is found in high levels within the cytoplasm of erythrocytes (5.6 mg/ml red blood cells) and yet less than 1% of the protein located in the cytoplasm is required to bind to the membrane in order to initiate the potassium efflux. The analysis of rat organ homogenates demonstrated that this protein is located in most tissues with particular enrichment in adrenal glands, brain, lung, and blood. These results demonstrate that there is a cytoplasmic protein, herein named calpromotin, which is a necessary and sufficient cytoplasmic component of calcium-dependent potassium transport in erythrocytes and perhaps other tissues.

Purification and measurement of calpromotin, the cytoplasmic protein which activates calcium-dependent potassium transport.

A simple procedure is described for the purification of calpromotin, a protein from the cytoplasm of red blood cells which is capable of activating calcium-dependent potassium transport. The purification steps involve a salt gradient elution from an anion exchange column (Whatman DE-52) followed by a potassium phosphate gradient elution from a column of hydroxyapatite (HA Ultrogel). These steps result in a 54% yield with a 161 fold purification. The calpromotin is estimated to be 99% pure as determined by densitometry of the protein profile on an SDS polyacrylamide gel. A competitive enzyme-linked immunosorbent assay (ELISA) using rabbit anti-human calpromotin antibodies, is described for measuring levels of calpromotin in the 5 to 100 ng range.