ERA

Download the full-sized PDF of Post-translational myristoylation during cell deathDownload the full-sized PDF

Analytics

Share

Permanent link (DOI): https://doi.org/10.7939/R36X2S

Download

Export to: EndNote  |  Zotero  |  Mendeley

Communities

This file is in the following communities:

Graduate Studies and Research, Faculty of

Collections

This file is in the following collections:

Theses and Dissertations

Post-translational myristoylation during cell death Open Access

Descriptions

Other title
Subject/Keyword
chemical biology
myristoylation
PKCepsilon
apoptosis
Huntingtin
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Martin, Dale David Orr
Supervisor and department
Berthiaume, Luc (Cell Biology)
Examining committee member and department
Robbins, Stephen (Department of Oncology at University of Calgary)
Barry, Michele (Medical microbiology and immunology)
Lehner, Richard (Departments of Pediatrics and Cell Biology)
Simmen, Thomas (Cell Biology)
Department
Department of Cell Biology
Specialization

Date accepted
2012-05-31T14:17:07Z
Graduation date
2011-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R36X2S
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Martin, Dale (2011) Biochimie  http://www.sciencedirect.com/science/article/pii/S0300908410003780Yap
, Megan (2010) J. Lipid. Res.  http://http://www.jlr.org/content/51/6/1566Martin
, Dale (2008) FASEB J  http://www.fasebj.org/content/22/3/797.long

File Details

Date Uploaded
Date Modified
2014-04-29T15:21:37.090+00:00
Audit Status
Audits have not yet been run on this file.
Characterization
File format: pdf (Portable Document Format)
Mime type: application/pdf
File size: 6575221
Last modified: 2015:10:12 10:34:13-06:00
Filename: Martin_Dale_Fall 2011.pdf
Original checksum: 71a1dd9ef79e911763f51b4897a7f3a8
Well formed: true
Valid: true
Status message: Too many fonts to report; some fonts omitted. Total fonts = 1334
File author: Dale's Beast
Page count: 314
File language: en-CA
Activity of users you follow
User Activity Date