The tumor suppressor protein p27 is often deregulated in aggressive human cancers via post-transcriptional mechanisms. Identifying and understanding how this occurs is essential for developing effective therapies to treat these types of tumors. Because p27 an unstructured and heat stable protein, we postulated its activity might be modulated by enzymes imparting structure to its polypeptide chain. Intriguingly, some cancers exhibit elevated levels of transglutaminase, a family of enzymes best known for cross linking proteins via formation of an isopeptide bond between glutamine and lysine residues on different protein monomers. To determine whether 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. Surprisingly, p27 monomers were not crosslinked as expected. Instead, p27 migrated faster on the gel, suggesting a more compact form of the monomer. P27 structure was evaluated by limited protease digest and supported a change in p27 confirmation. Importantly, the related family member p21 was not modified, suggesting the reaction was specific to p27. To determine if altering p27 structure modified its function, we analyzed the effect on p27 binding partners. Results indicate that transglutaminase can alter p27 structure and function, which might represent a novel regulatory pathway involved in the development of cancerous tumors.

Effect of transglutaminase on the deregulation of the p27 protein

249th National Meeting (2015)

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