The structure of alpha 2-macroglobulin-methylamine after papain digestion as determined by electron microscopy.

alpha 2-Macroglobulin-methylamine (alpha 2M-CH3NH2) was digested with papain at pH 5.0. The major 600 kDa fragment was purified by molecular-exclusion chromatography. In a non-denaturing gel-electrophoresis system, the 600 kDa fragment migrated in a single band at a rate that was comparable with that for the untreated alpha 2M-CH3NH2. The elution volume of the 600 kDa fragment on Superose-6 was slightly increased. In primary cultures of rat hepatocytes, cellular uptake of 125I-alpha 2M-CH3NH2 was not affected by the 600 kDa fragment, confirming the results of other investigators. The 600 kDa fragment was negatively stained with uranyl formate and analysed by transmission electron microscopy. The major structural characteristics of the parent protein (alpha 2M-CH3NH2) remained intact. The most common image included prominent lateral walls and two centrally located regions of stain exclusion termed 'paddle structures'. The distance between the paddle structures was equivalent in alpha 2M-CH3NH2 and the 600 kDa fragment [approximately 13.5 nm (135 A)]. By contrast, the lateral walls in the 600 kDa fragment were decreased in length by approximately 0.37 nm (37 A) (19%). It is proposed that the 600 kDa structure retains the 'hollow cylinder' shape of alpha 2M-CH3NH2. The structure of the cylinder is formed by the lateral walls and four paddle structures (only two are imaged, owing to overlapping). The paddle structures in the 600 kDa fragment are intact and relatively closer to the apices of the molecule, owing to the decrease in lateral wall length. Since the alpha 2M receptor-binding sites are removed by papain digestion, the studies presented here support the location of the receptor-binding sites near the apices of the lateral walls.     

Covalent complexes of albumin with serotonin, ketanserin and lysergic acid antagonize the activity of serotonin in human platelets

Covalent conjugates of bovine serum albumin (BSA) and 5-HT, ketanserin or d-lysergic acid were synthesized and characterized by polyacrylamide gel electrophoresis, whole blood clearance experiments in mice and aggregation studies with human platelets. Using the standard synthesis procedure, each mol of BSA bound 13.4 mol of [3H]5-HT. Derivatization did not cause significant protein aggregation as determined by electrophoresis. All three conjugates antagonized the ability of 5-HT to amplify aggregation caused by low concentrations of ADP. The antagonist activity of each conjugate was concentration dependent; 2.6 microM 5-HT-BSA completely inhibited the aggregation caused by 13 microM 5-HT. None of the BSA drug conjugates, including 5-HT-BSA, amplified platelet aggregation caused by ADP in the absence of 5-HT. Aggregation by ristocetin, collagen, epinephrine or ADP alone was not significantly affected by the conjugates. Whole blood elimination experiments in mice demonstrated that the three conjugates and underivatized BSA are equally stable in the circulation. These prototypic 5-HT drug-protein conjugates may be useful for probing 5-HT2 receptor-ligand interactions in human platelets.     

Electron microscopy studies of alpha 2-macroglobulin conformational intermediates obtained by derivatization with cis-dichlorodiammineplatinum (II)

The structure of human alpha 2-macroglobulin (alpha 2M) after reaction with cis-dichlorodiammineplatinum (II) (cis-DDP) was studied by electron microscopy. The cis-DDP stabilized a novel conformation of the native inhibitor resembling a doughnut surrounded by two, three, of four well defined spherules. When only two spherules were present, these structures were usually oriented on opposite sides of the doughnut. The protein region joining a spherule to the central structure did not include sufficient mass to exclude stain and was, therefore, invisible. Other images showed spherules that were partially superimposed on the doughnut. A comparison of many molecules suggested great flexibility of the peripheral spherules relative to the central structure. The cis-DDP prevented complete conformational change when the alpha 2M was reacted with trypsin. The products of this reaction included apparent conformational intermediates. These intermediates most closely resembled either native alpha 2M or the well established "H" structure of alpha 2M-proteinase, depending on the initial conditions used to modify the alpha 2 M with cis-DDP. When cis-DDP-treated alpha 2M was reacted with trypsin, purified by chromatography and subsequently treated with diethyldithiocarbamate, complete conformational change was observed. Based on an analysis of the alpha 2M structural intermediates obtained using the chemical modification procedures described here, a new model of alpha 2M conformational change was developed. We postulate that conformational change initially involves contraction of the peripheral spherules towards the central doughnut. These spherules then unfold and elongate in the perpendicular direction to form the lateral walls of the proteinase transformed alpha 2M H structure.     

Molecular dissection of the human alpha2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling

alpha(2)-Macroglobulin (alpha(2)M) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in alpha(2)M are responsible for its various functions, we hypothesized that the overall effects of alpha(2)M on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). FP6 rapidly and robustly activated Akt and ERK/MAP kinase in Schwann cells and PC12 cells. This response was blocked by LRP-1 gene silencing or by co-incubation with the LRP-1 antagonist, receptor-associated protein. The activity of FP6 also was blocked by mutating Lys(1370) and Lys(1374), which precludes LRP-1 binding. FP3 blocked activation of Akt and ERK/MAP kinase in response to nerve growth factor-beta (NGF-beta) but not FP6. In PC12 cells, FP6 promoted neurite outgrowth and expression of growth-associated protein-43, whereas FP3 antagonized the same responses when NGF-beta was added. The ability of FP6 to trigger LRP-1-dependent cell signaling in PC12 cells was reproduced by the 18-kDa RBD, isolated from plasma-purified alpha(2)M by proteolysis and chromatography. We propose that the effects of intact alpha(2)M on cell physiology reflect the degree of penetration of activities associated with different domains, such as FP3 and FP6, which may be regulated asynchronously by conformational change and by other regulatory proteins in the cellular microenvironment.     

Erythropoietin promotes MCF-7 breast cancer cell migration by an ERK/mitogen-activated protein kinase-dependent pathway and is primarily responsible for the increase in migration observed in hypoxia

Recent studies indicate that cancer cells express erythropoietin receptor (EpoR). In this study, we have shown that erythropoietin (Epo) activates the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and promotes migration in MCF-7 breast cancer cells. Epo-stimulated MCF-7 cell migration was blocked by the MEK inhibitor PD098059 and by dominant negative MEK-1, indicating an essential role for ERK. When MCF-7 cells were exposed to hypoxia (1.0% O(2)) for 3 h, the Epo mRNA level increased 2.4 +/- 0.5-fold, the basal level of ERK activation increased, and cell migration increased 2.0 +/- 0.1-fold. Soluble EpoR and Epo-neutralizing antibody significantly inhibited hypoxia-induced MCF-7 cell migration, suggesting a major role for autocrine EpoR cell signaling. MCF-7 cell migration under hypoxic conditions was also inhibited by PD098059. These experiments identify a novel pathway by which exogenously administered Epo, and Epo that is produced locally by cancer cells under hypoxic conditions, may stimulate cancer cell migration.     

Soluble urokinase-type plasminogen activator receptor inhibits cancer cell growth and invasion by direct urokinase-independent effects on cell signaling

The urokinase-type plasminogen activator receptor (uPAR) is released from human cancers and is readily detected in blood. In animal models, soluble uPAR (SuPAR) antagonizes cancer progression; however, the mechanism by which SuPAR functions in vivo remains unclear. It is generally thought that SuPAR scavenges uPA and prevents its interaction with membrane-anchored uPAR. In this study, we demonstrate a novel molecular mechanism by which SuPAR may inhibit cancer progression. We show that SuPAR has the potential to directly and in a uPA-independent manner block the signaling activity of membrane-anchored uPAR. Whether SuPAR inhibits signaling is cell type-specific, depending on the state of the endogenous uPA-uPAR signaling system. In uPAR-deficient cells that lack endogenous uPAR signaling, including uPAR-/-murine embryonic fibroblasts and human embryonal kidney 293 cells, SuPAR functions as a partial signaling agonist that activates ERK/mitogen-activated protein kinase. By contrast, in cells with potent autocrine uPA-uPAR signaling systems, including MDA-MB 231 breast cancer cells and low density lipoprotein receptor-related protein-1-deficient murine embryonic fibroblasts, SuPAR substantially decreases ERK activation. The mechanism probably involves competitive displacement of membrane-anchored uPAR-uPA complex from signaling adaptor proteins. As a result of its effects on cell signaling, SuPAR blocks cell growth and inhibits cellular invasion of Matrigel. Cleavage of SuPAR by proteinases increases its signaling agonist activity and reverses its inhibitory effects on growth and invasion. Thus, proteolytic cleavage represents a molecular switch that neutralizes the anticancer activity of SuPAR.     

Epidermal growth factor receptor-dependent and -independent cell-signaling pathways originating from the urokinase receptor

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

Improving the specificity of high-throughput ortholog prediction

Background  

Orthologs (genes that have diverged after a speciation event) tend to have similar function, and so their prediction has become an important component of comparative genomics and genome annotation. The gold standard phylogenetic analysis approach of comparing available organismal phylogeny to gene phylogeny is not easily automated for genome-wide analysis; therefore, ortholog prediction for large genome-scale datasets is typically performed using a reciprocal-best-BLAST-hits (RBH) approach. One problem with RBH is that it will incorrectly predict a paralog as an ortholog when incomplete genome sequences or gene loss is involved. In addition, there is an increasing interest in identifying orthologs most likely to have retained similar function.     

Plasminogen carbohydrate side chains in receptor binding and enzyme activation: a study of C6 glioma cells and primary cultures of rat hepatocytes.

The human [Glu1]-plasminogen carbohydrate isozymes, plasminogen type I (Pg 1) and plasminogen type II (Pg 2), were separated by chromatography and studied in cell binding experiments at 4 degrees C with primary cultures of rat hepatocytes and rat C6 glioma cells. In both cell systems, Pg 1 and Pg 2 bound to an equivalent number of receptors, apparently representing the same population of surface molecules. The affinity for Pg 2 was slightly higher. With hepatocytes, the KD for Pg 1 was 3.2 +/- 0.2 microM, and the KD for Pg 2 was 1.9 +/- 0.1 microM, as determined from Scatchard transformations of the binding isotherms. The Bmax was approximately the same for both isozymes. With C6 cells, the KD for Pg 1 was 2.2 +/- 0.1 microM vs. 1.5 +/- 0.2 microM for Pg 2. Again, the Bmax was similar with both isozymes. 125I-Pg 1 and 125I-Pg 2 were displaced from specific binding sites by either nonradiolabeled isozyme. The KI for Pg 2 was slightly lower than the KI for Pg 1 with hepatocytes (0.9 vs. 1.3 microM) and with C6 cells (0.6 vs. 1.1 microM). No displacement was detected with miniplasminogen at concentrations up to 5.0 microM. Activation of Pg 1 and Pg 2 by recombinant two-chain tissue-plasminogen activator (rt-PA) was enhanced by hepatocyte cultures. The enhancing effect was greater with Pg 2. Hepatocyte cultures did not affect the activation of miniplasminogen by rt-PA or the activation of plasminogen by streptokinase. Unlike the hepatocytes, C6 cells did not enhance the activation of plasminogen by rt-PA or streptokinase; however, plasmin generated in the presence of C6 cells reacted less readily with alpha 2-antiplasmin.