The tumor suppressor protein p27 is often deregulated in aggressive human cancers via post-transcriptional mechanisms. Understanding the mechanism by which this occurs is essential for developing therapies to treat these tumors. Interestingly, some cancerous tumors express elevated levels of transglutaminase, a family of enzymes which cross link proteins via the formation of an isopeptide bond between glutamine and lysine groups. In order to observe whether or not transglutaminase could alter p27 structure, we purified His tagged p27 from E. coli by nickel affinity chromatography and incubated it with transglutaminase. Products were then analyzed by denaturing polyacrylamide gel electrophoresis. In a surprising outcome, it was observed that p27 monomers were not cross-linked as expected. Instead, p27 migrated faster on the gel, which suggests that a more compact form of the monomer was formed. Two fragments of p27, one with amino acids 1-86 and the other with amino acids 87-198, were incubated individually with transglutaminase in order to narrow down the specific glutamine and lysine residues involved in the formation of the isopeptide bond. The results showed that the groups involved in the formation of the bond were located on the C-terminus fragment (amino acids 87-198), whereas the N-terminus fragment did not show any changes in conformation.

Enzymatic modification of p27kip1 by transglutaminase

251st National Meeting (2016)

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