Post-translational myristoylation during cell death

• Author / Creator

  Martin, Dale David Orr

• Myristoylation involves the addition of a 14-carbon fatty acid to the N-terminal glycine of proteins by N-myristoyltransferase. Myristoylation promotes protein-membrane and protein-protein interactions that are crucial for the function of myristoylated proteins. Myristoylation occurs co-translationally on the nascent polypeptide following the removal of the initiator methionine or post-translationally following the proteolytic exposure of an N-terminal glycine.
  Herein, we describe how we employed bio-orthogonal myristate analogs that are incorporated into proteins at N-terminal glycines in an N-myristoyltransferase dependent manner, and subsequently, are chemoselectively ligated to various affinity tags that allowed the facile detection of the myristoylated proteins. These methods allowed the detection of myristoylated proteins by western blotting with exposure times ranging from seconds to minutes (~1-5 million faster compared to the incorporation of radioactive myristate into proteins).
  Ultimately, we identified 7 post-translationally myristoylated proteins during apoptosis. These include the following caspase cleaved protein products: cell division control protein 6 homolog, cytoplasmic dynein-intermediate chain 2A, Huntingtin (Htt), microtubule-actin crosslinking factor 1, the apoptotic regulator induced myeloid leukemia cell differentiation protein, protein kinase C epsilon (PKCε) and isoform 1 of YTH domain family protein 2.
  Furthermore myristoylated ctPKCε was found to localize to membranes, increase ERK signaling and degradation of the pro-apoptotic protein Bim, which prevented a significant loss of mitochondrial potential of 17% over non-myristoylated ctPKCε in HeLa cells in the presence of apoptotic stimuli. Together, this suggests a possible anti-apoptotic role for post-translationally myristoylated caspase cleaved ctPKCε.
  In addition, a 34 amino acid post-translationally myristoylated fragment of Htt released by the cleavage of two caspases was found to localize to ER and lysosomes. Moreover, overexpression of myr-ctHttN34-EGFP induced the formation of autophagosome-like vesicles that were associated with cell death in HeLa cells.
  Overall, the new tools described within will enable the field of myristoylation by providing methods to rapidly detect, identify and characterize myristoylated proteins. Already, it has nearly tripled the number of identified post-translationally myristoylated proteins and through the identification of myr-ctHtt and myr-ctPKCε, we have shown the relevance of post-translational myristoylation during apoptosis with plausible implications in the pathophysiology of Huntington’s disease.

• Subjects / Keywords

  • PKCepsilon
  • Huntingtin
  • Apoptosis
  • Myristoylation
  • Chemical biology

• Graduation date

  Fall 2011

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R36X2S

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.