BACKGROUND: Tissue factor, which is required for the initiation of the extrinsic coagulation cascade, is known to be upregulated in cells within atherosclerotic lesions, including smooth muscle cells. Tissue factor expression on the smooth muscle cell surface could be of pathological significance as a contributor to plaque growth, thrombus formation, and the acute coronary syndrome after plaque rupture. METHODS AND RESULTS: In this study, we show that LDL increased tissue factor mRNA and cell surface protein in smooth muscle cells without a marked increase in surface tissue factor activity. Hydrogen peroxide activated tissue factor on the cell surface but did not increase tissue factor mRNA or cell surface protein. Sequentially added LDL and hydrogen peroxide increased mRNA, cell surface protein, and activity; surface activity was greater than that observed with hydrogen peroxide alone. The action of hydrogen peroxide did not involve a regulatory mechanism associated with the cytoplasmic tail of tissue factor because a truncated tissue factor lacking the cytoplasmic tail was activated by hydrogen peroxide. CONCLUSIONS: These results suggest a novel 2-step pathway for increased tissue factor activity on smooth muscle cell surfaces in which lipoproteins regulate synthesis of a latent tissue factor and oxidants activate the protein complex. (+info)
Inhibition of clathrin-coated pit assembly by an Eps15 mutant.
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Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits. (+info)
HP33: hepatocellular carcinoma-enriched 33-kDa protein with similarity to mitochondrial N-acyltransferase but localized in a microtubule-dependent manner at the centrosome.
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Using a new subtraction method and chemically induced rat hepatocellular carcinomas, we identified a hepatocellular carcinogenesis and hepatocyte proliferation-related gene designated hp33 that encoded a 33-kDa protein. The predicted protein was similar to the bovine aralkyl N-acyltransferase and arylacetyl N-acyltransferase. HP33 was restrictively expressed in the liver and kidney, and its gene expression was stimulated in the regenerating liver as well as in hepatocellular carcinoma. Interestingly, it was demonstrated in various hepatic cells that HP33 was localized in regions surrounding the centrosome, where mitochondria were not concentrated. Moreover, its centrosomal localization was evident in the interphase but not in the mitotic phase of the cell cycle. The centrosomal localization of HP33 was dependent on microtubules, and ectopically expressed HP33 was seen at centrosomes even in fibroblasts, which do not exhibit a typical staining pattern of HP33. The centrosomal localization of HP33 became invisible by nocodazole treatment, whereas the mitochondrial staining pattern was not affected by it. In vitro cosedimentation experiments using purified microtubules indicated that HP33 bound to MTs directly and that its MT-binding ability was dependent on the C-terminal basic domain of the protein. These results suggest that, different from early predictions based on its primary structure, HP33 has a growth- and carcinogenesis-related function that may be independent of mitochondrial function. (+info)
Heteropolymerization of S, I, and Z alpha1-antitrypsin and liver cirrhosis.
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The association between Z alpha1-antitrypsin deficiency and juvenile cirrhosis is well-recognized, and there is now convincing evidence that the hepatic inclusions are the result of entangled polymers of mutant Z alpha1-antitrypsin. Four percent of the northern European Caucasian population are heterozygotes for the Z variant, but even more common is S alpha1-antitrypsin, which is found in up to 28% of southern Europeans. The S variant is known to have an increased susceptibility to polymerization, although this is marginal compared with the more conformationally unstable Z variant. There has been speculation that the two may interact to produce cirrhosis, but this has never been demonstrated experimentally. This hypothesis was raised again by the observation reported here of a mixed heterozygote for Z alpha1-antitrypsin and another conformationally unstable variant (I alpha1-antitrypsin; 39Arg-->Cys) identified in a 34-year-old man with cirrhosis related to alpha1-antitrypsin deficiency. The conformational stability of the I variant has been characterized, and we have used fluorescence resonance energy transfer to demonstrate the formation of heteropolymers between S and Z alpha1-antitrypsin. Taken together, these results indicate that not only may mixed variants form heteropolymers, but that this can causally lead to the development of cirrhosis. (+info)
A three-dimensional construction of the active site (region 507-749) of human neutral endopeptidase (EC.3.4.24.11).
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A three-dimensional model of the 507-749 region of neutral endopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integrating the results of secondary structure predictions and sequence homologies with the bacterial endopeptidase thermolysin. Additional data were extracted from the structure of two other metalloproteases, astacin and stromelysin. The resulting model accounts for the main biological properties of NEP and has been used to describe the environment close to the zinc atom defining the catalytic site. The analysis of several thiol inhibitors, complexed in the model active site, revealed the presence of a large hydrophobic pocket at the S1' subsite level. This is supported by the nature of the constitutive amino acids. The computed energies of bound inhibitors correspond with the relative affinities of the stereoisomers of benzofused macrocycle derivatives of thiorphan. The model could be used to facilitate the design of new NEP inhibitors, as illustrated in the paper. (+info)
Evolution of a protein fold in vitro.
(38/55031)
A "switch" mutant of the Arc repressor homodimer was constructed by interchanging the sequence positions of a hydrophobic core residue, leucine 12, and an adjacent surface polar residue, asparagine 11, in each strand of an intersubunit beta sheet. The mutant protein adopts a fold in which each beta strand is replaced by a right-handed helix and side chains in this region undergo significant repacking. The observed structural changes allow the protein to maintain solvent exposure of polar side chains and optimal burial of hydrophobic side chains. These results suggest that new protein folds can evolve from existing folds without drastic or large-scale mutagenesis. (+info)
The C-D interhelical domain of the serpin plasminogen activator inhibitor-type 2 is required for protection from TNF-alpha induced apoptosis.
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The serine proteinase inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), has been reported to inhibit tumor necrosis factor-alpha (TNF) induced apoptosis. In order to begin to understand the molecular basis for this protection, we have investigated the importance of a structural domain within the PAI-2 molecule, the C-D interhelical region, in mediating the protective effect. The C-D interhelical region is a 33 amino acid insertion which is unique among serpins and has been implicated in transglutaminase catalyzed cross-linking of PAI-2 to cell membranes. We have constructed a mutant of PAI-2 wherein 23 amino acids are deleted from the C-D interhelical region generating a structure predicted to be homologous to the closely related, but non-inhibitory serpin, chicken ovalbumin. The PAI-2Delta65/87 deletion mutant retained inhibitory activity against its known serine proteinase target, urokinase-type plasminogen activator (uPA); however expression of this mutant in HeLa cells failed to protect from TNF-induced apoptosis. Analyses of the cellular distribution of PAI-2 showed that intracellular PAI-2, and not secreted or cell-surface PAI-2, was likely responsible for the observed protection from TNF-induced apoptosis. No evidence was found for specific cross-linking of PAI-2 to the plasma membrane in either control or TNF/cycloheximide treated cells. The data demonstrate that the PAI-2 C-D interhelical domain is functionally important in PAI-2 protection from TNF induced apoptosis and suggest a novel function for the C-D interhelical domain in the protective mechanism. (+info)
Induction of albuminuria in mice: synergistic effect of two monoclonal antibodies directed to different domains of aminopeptidase A.
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BACKGROUND: Aminopeptidase A is an enzyme that is present on podocytes and is involved in the degradation of angiotensin II. In previous studies in mice, we administered single monoclonal antibodies directed against aminopeptidase A. We observed that only monoclonal antibodies that inhibited aminopeptidase A enzyme activity caused albuminuria. METHODS: In this study, the effects of the combined injections of two monoclonal anti-aminopeptidase A antibodies (mAbs) were studied, using a combination of anti-aminopeptidase A mAbs that were directed against two different domains involved in the aminopeptidase A enzyme activity (ASD-3 or ASD-37) and an anti-aminopeptidase A mAb not related to the enzyme active site (ASD-41). RESULTS: An injection of the combinations ASD-3/37 (total 4 mg, 1:1 ratio) and ASD-37/41 (total 4 mg, 1:1 ratio) in doses that do not cause albuminuria when given alone (4 mg) induced massive albuminuria at day 1 after injection. The combination ASD-3/41 had no effect. This albuminuria was not dependent on systemic immune mediators of inflammation and could not merely be related to a blockade of aminopeptidase A enzyme activity. However, a correlation was observed between the induction of albuminuria and the aggregation of the mAbs injected and aminopeptidase A on the podocytes. An injection of the combinations ASD-3/37 or ASD-37/41 did not cause an increase in systemic blood pressure. The treatment with a combination of enalapril and losartan lowered blood pressure (53 +/- 10 vs. 90 +/- 3 mm Hg in untreated mice) and reduced the acute albuminuria by 55% (11,145 +/- 864 vs. 24,517 +/- 2448 micrograms albumin/18 hr in untreated mice). However, similar effects were observed using triple therapy. Therefore, the reduction of albuminuria by the combined treatment of enalapril/losartan seems to be the consequence of the reduction in the systemic blood pressure. These findings argue against a specific role for angiotensin II in this model. CONCLUSIONS: The combined injection of two mAbs directed against different domains of aminopeptidase A induces a massive albuminuria in mice, which is not merely dependent on angiotensin II. We hypothesize that the direct binding of mAbs to at least two pathogenic domains on aminopeptidase A triggers the podocyte to release mediators that are involved in the observed albuminuria. (+info)