Supplementary Information Video attached / <p> Machado-Joseph disease (MJD), also known as Spinocerebellar ataxia type 3
(SCA3) is one of nine poly glutamine neurodegenerative diseases caused by an expansion of CAG DNA triplets in the genes resulting in an expanded poly glutamine tract in the expressed proteins. These proteins are unrelated in function yet all manifest as specific neurological diseases. The Truant lab and others have previously shown that six of the nine polyglutamine proteins display nucleocytoplasmic shuttling capabilities and that this shuttling is affected by polyglutamine expansion. It is believed that deciphering the mechanism of nucleocytoplasmic transport may be important in understanding the normal function of these proteins, which in turn may lead to a better understanding of the pathogenesis of disease. Studies that looked at the subcellular localization of the MJD/SCA3 protein, ataxin-3, have shown that the normal protein is variably distributed between the nucleus and the cytoplasm, whereas mutant ataxin-3 is localized primarily in the nucleus. Using fluorescent protein technology and fluorescence microscopy, this thesis project attempts to analyze the nucleocytoplasmic shuttling capabilities of ataxin-3 and to evaluate the potential mechanisms that govern its translocation into and out of the nucleus. </p> <p> It was revealed that ataxin-3 is able to shuttle into and out of the nucleus and that the shuttling dynamics are dependent on the length of the poly glutamine tract. As well, two putative, CRMl dependent nuclear export signals and a putative, importin-a/~1 dependent, classical, nuclear localization signal were tested and shown to be nonfunctional as transport signals. It was then discovered that ataxin-3 is marginally leptomycin B (an inhibitor ofCRMl dependent nuclear export) sensitive in NIH3T3 and MCF7 cells, more sensitive to the drug in STHdhQ71Q7 cells and even more so in HEK 293 cells. This suggests that an exogenous factor mediates the nuclear import of ataxin-3 through the CRMl pathway. Subsequently, four known binding partners, hHDACl, hHDAC2, hHDAC6 and hHRAD23b, were tested for their potential ability to shuttle ataxin-3. It was concluded that although hHDAC6 had the greatest effect on ataxin-3 subcellular localization, we believe that it does not mediate its nuclear import or export. Future studies would involve an investigation as to how and why different polyglutamine lengths affect the nucleocytoplasmic shuttling of ataxin-3 and to identify the factor(s) that cause ataxin-3 to be more sensitive to LMB treatments in HEK 293 cells. </p> / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21639 |
| Date | 11 1900 |
| Creators | Pinchev, Deborah |
| Contributors | Truant, Ray, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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