Acid-activatable Cysteine Proteinases in the Cellular Slime Mold

Abstract

Studies of the cysteine proteinases of the cellular slime mold Dictyostelium discoideum have been aided by a simple acid treatment step that was incorporated into the standard one-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay procedure. The step involved immersing the separating gel in 10% (v/v) glacial acetic acid for 30-60 s immediately after electrophoresis. This modified approach revealed the presence of acid-activatable forms of some enzymes with noticeable increases in their ability to hydrolyze gelatin, a substrate present in the sodium dodecyl sulfate-polyacrylamide gels, and peptidyl amidomethylcoumarins. The activation has been analyzed using extracts of dormant spores from which cysteine proteinase activity had previously appeared low or virtually absent. The major acid-activatable proteinase had an apparent molecular mass of 48 kDa. Its activation was not due to autocatalysis as it was not prevented by mercuric chloride, an inhibitor of the enzyme, and was not accompanied by a significant change in electrophoretic mobility. It was most likely due to a conformational change and/or the removal of a low molecular weight inhibitor. The acid treatment has also revealed the presence of acid-activatable cysteine proteinases in vegetative cells, in which cysteine proteinase activity is present at high levels, as well as among enzymes from the developmental cells which have much lower cysteine proteinase activity. Indeed novel developmental forms were detected at some stages. These results provide additional insight concerning cysteine proteinase expression at various stages during development in the slime molds. A developmental model is presented which suggests that the crypticity of the cysteine proteinases in dormant spores may be governed by proton pumps and endogenous lysosomotropic agents.

Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium

“…L. pneumophila Corby may inhibit proteolytic processing events which are required for proper sorting and maturing of lysosomal enzymes by inducing the upregulation of the Dictyostelium cysteine protease inhibitor CpiA expression (Richardson et al, 1988;North et al, 1996). The observation that Legionella phagosomes avoid the phagolysosome formation only at an early stage of infection supports our hypothesis since the increase of the cysteine protease inhibitor was only observed until the 2-h time point post-infection.…”

Transcriptome changes during fruit development and ripening of sweet orange (Citrus sinensis)

“…Some examples of this large cluster included genes encoding Cu/Zn superoxide dismutase ( TC12069 ), ascorbate peroxidase ( EY685405 , TC22775 and TC14669 ), MYB1 ( EY649968 ) and plastidic glucose 6-phosphate/phosphate translocator ( EY722703 ). Only five genes were detected as differentially expressed at all four stages; one encoded a cysteine protease (Cp5) ( TC5370 ), which had been reported exhibiting the acid-activatable cysteine protease forms [30], three had no assigned functions, and one shared no homology with any entry in. The five genes could be classified into two groups (two genes in one group, three genes in the other), with their expression patterns of being opposite to each other but with both groups having a turning point at 150 DAF (additional file 10).…”

Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat

“…The transient appearance of the MAb 4A11 epitope might be related to global disulfide rearrangements (34) or other posttranslational modifications such as proteolytic processing (32,39) thought to occur during coat assembly. SP65 may be associated with the outer coat surface because the GFP of SP65-GFP is accessible at the spore surface based on Ab labeling and is likely exposed to proteases present within the spore coat and the interspore matrix (10,19,32). Indeed, SP65 is susceptible to proteolytic degradation when expressed in the growth medium (Fig.…”