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Interleukin-like EMT inducer (ILEI,FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell-biological process that confers metastatic properties to a tumor cell. However, very little is known about how ILEI is regulated. Here we demonstrate that ILEI is an in vivo regulator of melanoma invasiveness and is transcriptionally up-regulated by the upstream stimulatory factor-1 (USF-1), an E-box--binding, basic-helix-loop-helix family transcription factor. shRNA-mediated knockdown of ILEI in melanoma cell lines attenuated lung colonization but not primary tumor formation. We also identified the mechanism underlying ILEI transcriptional regulation, which was through a direct interaction of USF-1 with the ILEI promoter. Of note, stimulation of endogenous USF-1 by UV-mediated activation increased ILEI expression, whereas shRNA-mediated USF-1 knockdown decreased ILEI gene transcription. Finally, we report thatknockingdownUSF-1decreasestumorcellmigration.Insummary, our work reveals that ILEI contributes to melanoma cell invasiveness in vivo without affecting primary tumor growth and is transcriptionally up-regulated by USF-1. [ABSTRACT FROM AUTHOR]

Aerolysin is a bacterial toxin that binds to glycosyiphosphatidylinositol-anchored proteins (GPI-AP) on mammalian cells and oligomerizes, inserting into the target membranes and forming channels that cause cell death. We have made a variant of aerolysin, R336A, that has greatly reduced the ability to bind to GPI-AP, and as a result it is only very weakly active. Fusion of interleukin 2 (1L2) to the N terminus of R336A-aerolysin results in a hybrid that has little or no activity against cells that do not have an 1L2 receptor because it cannot bind to the GPI-AP on the cells. Strikingly, the presence of the 1L2 moiety allows this hybrid to bind to cells displaying high affinity 1L2 receptors. Once bound, the hybrid molecules form insertion-competent oligomers. Cell death occurs at picomolar concentrations of the hybrid, whereas the same cells are insensitive to much higher concentrations of R336A-aerolysin lacking the 1L2 domain. The targeted channel-forming hybrid protein may have important advantages as a therapeutic agent. [ABSTRACT FROM AUTHOR]

MUC5AC mucins secreted by HT-29 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, D. J. (2000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and dimer, GalNAc-substituted dimer, fully glycosylated dimer, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking approximately 2-4 h). The results show that the mucin polypeptide undergoes dimerization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin dimer, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.

Aeromonas sp. secrete a lipase/acyltransferase that shares several properties with the mammalian plasma enzyme lecithin:cholesterol acyltransferase. Reaction of the enzyme with tetranitromethane led to modification of 2 tyrosines and a nearly 80% decline in enzyme activity. Replacing Tyr230 with Phe altered the activity of the enzyme in the same way as did treatment with tetranitromethane. Unlike the wild type enzyme, which preferentially hydrolyzes the 2-position acyl chain of phosphatidylcholine, the Y230F mutant enzyme did not discriminate between the 1- and 2-positions of the phospholipid. Tyr230 may be necessary to correctly position phospholipid substrates at the active site. Several amino acids around the active site Ser16 of the lipase were also changed. Replacing Ser18 with Gly, bringing the enzyme's sequence into line with the "lipase consensus sequence," resulted in reduced secretion of the protein and complete loss of activity. Changing this serine to Val led to an inactive protein that was not secreted at all. Substituting Phe13 in the hydrophobic region of the consensus sequence with Ser also prevented secretion, although the mutant protein appeared to be active. The Aeromonas lipase may represent a distinct group of lipolytic enzymes which have a novel active site structure.

Aerolysin is a channel-forming protein secreted as a protoxin by Aeromonas hydrophila. Analytical centrifugation measurements showed that proaerolysin is a dimer in solution, and this was confirmed by chemical cross-linking with dimethyl suberimidate. Dissociation of proaerolysin with low concentrations of SDS resulted in the loss of tertiary structure, assessed by near ultraviolet circular dichroism. This was accompanied by an increase in the protein's ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonate, as well as by increased sensitivity to proteolytic degradation. However, the monomer was not fully unfolded by the detergent, as the tryptophans remained in a hydrophobic environment, and the secondary structure measured by far ultraviolet circular dichroism did not seem to be affected. Aerolysin, the active form of the protein, was also shown to be a dimer, and its stability was found to be no different from the stability of the protoxin dimer. Substituting tryptophan 371 or tryptophan 373 with leucine greatly reduced the stability of dimeric proaerolysin. These substitutions are known to increase the protein's ability to oligomerize, supporting the conclusion that dimer dissociation is necessary for oligomerization to occur.

The channel-forming protein aerolysin must cross both the inner and outer bacterial membranes during its secretion from Aeromonas hydrophila or from Aeromonas salmonicida containing the cloned structural gene. We examined the fate of three mutant proteins in which Trp-227, near the middle of the amino acid chain, was replaced with glycine, leucine, or phenylalanine by site-directed mutagenesis. All three proteins crossed the inner membrane and entered the periplasm in the same way as wild-type, and in each case the signal sequence was removed correctly. Little or none of the proaerolysin substituted with glycine or leucine was released into the culture supernatant. Instead, significant amounts became associated with the outer membrane. The Phe-227 protoxin was secreted by the bacteria but at a reduced rate. The leucine and phenylalanine mutant proteins were purified and compared with native proaerolysin. They were processed correctly to the mature forms by treatment with trypsin, and like native aerolysin, both were resistant to further proteolysis. In each case, processing was followed by the formation of oligomers similar to those produced by native toxin. The hemolytic activity of the processed Phe-227 mutant was one-quarter that of wild-type toxin whereas Leu-227 aerolysin had less than one-hundredth the wild-type activity. These results are further evidence that aerolysin is secreted in at least two steps. As well, they show that the last step, crossing the outer membrane, can be blocked by an apparently small change in the structure of the protein.