Mitochondria have evolved to play a vital role in both the life and death of a eukaryotic cell, through involvement in numerous cellular functions, such as the proficient production of energy from ATP biosynthesis and the regulation of programmed cell death. As a result, dysfunction in the biochemical processes housed within this organelle is implicated in diverse diseases, including cancer, diabetes, and neurodegenerative disorders. Advancing mitochondrial medicine by probing the subcellular biochemistry or targeting therapeutics into this organelle has motivated the development of effective mitochondrial delivery vectors. Thus, the rational design of novel mitochondrial-specific molecules, inspired by the success of cell-penetrating peptides, is described, whereby short synthetic peptides that retain the ability to traverse the plasma membrane, yet with mitochondrial-specificity were engineered. By modulating the overall physicochemical properties, through substitutions with both natural and synthetic amino acids, and monitoring the intracellular localization by confocal fluorescence microscopy, the requisite thresholds for achieving mitochondrial accumulation with a cationic peptide were elucidated. These systematic studies led to the development of a novel class of cationic yet lipophilic peptides, referred to as mitochondria-penetrating peptides (MPPs), which are readily cell permeable and preferentially localize into the mitochondria of living mammalian cells. The mechanisms of cellular uptake and mitochondrial matrix accumulation were investigated and the results from these studies suggest that MPPs utilize the negative membrane potential across these biological membranes to drive translocation. In addition, the effects of various chemical perturbations on the cellular and mitochondrial uptake, such as sequence, structure of the cation moiety, and chirality, were examined. The information obtained from these studies provided insight into the important features of these peptides and led to the design of an optimized molecule displaying pyridinium salt side chains. Moreover, MPPs were shown to be effective mitochondrial delivery vectors for diverse and bioactive small molecule cargo. In conclusion, the extensive biological and chemical characterization of MPPs revealed the importance of balancing the opposing characteristics of positive charge and lipophilicity to attain preferential sequestration into mitochondria, as well as provided evidence that these peptides will be suitable as mitochondrial delivery vectors.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/26328 |
| Date | 23 February 2011 |
| Creators | Stewart, Kelly M. |
| Contributors | Kelley, Shana O. |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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