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Further evidence for a common mechanism for shedding of cell surface proteins
J Müllberg1, C T Rauch, M F Wolfson
1Immunex Corporation, Seattle, WA 98101, USA. mullbergj@immunex.com
Abstract:
Pro-TNF alpha, Steel factor, type II IL-1R and IL-2R alpha were expressed in COS-7 cells and the generation of their soluble forms was examined. The release of all four proteins was strongly stimulated by the phorbol ester PMA and completely blocked by a hydroxamate-based inhibitor of metalloproteases. COS-7 cell membranes were found to cleave various synthetic pro-TNF alpha peptides with the same specificity as a partially purified TNF alpha converting enzyme purified from human monocytic cells, suggesting that the same enzyme may be responsible for at least some of the COS-7 cell shedding activity.
Insights
Researchers studied the shedding of soluble proteins like pro-tumor necrosis factor alpha (TNF alpha). Phorbol ester PMA stimulated release, while a metalloprotease inhibitor blocked it, suggesting enzyme involvement in protein shedding.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Soluble forms of certain proteins are generated through shedding.
- Tumor necrosis factor alpha (TNF alpha) and other growth factors have soluble counterparts.
Purpose of the Study:
- To investigate the mechanisms of soluble protein generation from COS-7 cells.
- To identify factors influencing the shedding of pro-tumor necrosis factor alpha (pro-TNF alpha), Steel factor, IL-1R type II, and IL-2R alpha.
Main Methods:
- Expression of pro-TNF alpha, Steel factor, IL-1R type II, and IL-2R alpha in COS-7 cells.
- Treatment with phorbol ester PMA to stimulate protein release.
- Inhibition studies using a hydroxamate-based metalloprotease inhibitor.
- Analysis of COS-7 cell membrane proteolytic activity on synthetic pro-TNF alpha peptides.
Main Results:
- The release of all four studied proteins was significantly enhanced by PMA.
- A hydroxamate-based metalloprotease inhibitor completely blocked the shedding of these proteins.
- COS-7 cell membranes exhibited proteolytic cleavage of pro-TNF alpha peptides with specificity similar to a known TNF alpha converting enzyme.
Conclusions:
- The shedding of pro-TNF alpha, Steel factor, IL-1R type II, and IL-2R alpha from COS-7 cells is mediated by metalloproteases.
- The enzyme responsible for shedding in COS-7 cells may be similar or identical to the TNF alpha converting enzyme found in human cells.