Question

Describe the two-part activation mechanism for TACE. What is the role of PMA and PKC in this activation?

9. Name another APP cleavage product we could have analyzed to test our hypothesis. What would you predict would happen to levels of this product following PMA treatment?

Answer 1

TACE mediated ectodomain cleavage is activated by signalling of the MAPKs p38 and ERK. Upon activation of the ERK and p38 MAPKs pathway, the balance shifted from TACE dimers to monomers and this shift was associated with increased cell surface presentation of TACE and decreased TIMP3 association, which releaved the inhibition of TACE by TIMP3 and increased TACE mediated proteolysis of transforming growth factor . Thus, cell signalling altered the dimer monomer equilibrium and inhibitor association of promote activation of TACE mediated ectodomain shedding. PMA induces both reactive ROC generation and and TNF p75 receptor shedding in mono mac 6 Cells, a human monocytic cell lines and I selecting shedding in hurkat t-cells. PMA binds to activate PKC which results, in changing shedding conformation or promoting sheddase/substrate interactions, which leads to enhance ectodomain shedding.

9. Ans- The CTF fragments generated by alpha and beta secretase are called CTF83 and CTF99 will result in the generation of p3 and A-beta as well as the AICD. A multi protein complex constitutes gamma secreatase activity , four proteins are minimally required for this comex are PS1, Nct, pen2 and Aph1. If beta-secreatase were limited by the availability of APP, then incresed cleavage of APP by other secretases could decrease beta secretase clevage of APP and hence A-beta production. To determine whether or not to increased alpha reg lead to reduce beta secretase activity, the effects of PMA induced CTF activity on beta-secretion of APP, using an antibody that specifically recognises sAPP beta for immunoprecipitation. PMA stimulation of APP alpha-secretase cleavage causes decreased beta secretase clevage due to metalloprotease activation.