Date of Award

5-9-2024

Publication Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

Keywords

Cancer;Cell cycle;Tuberous Sclerosis Complex

Supervisor

Lisa Porter

Abstract

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder that occurs in approximately 1 in 6000 live births. The disorder is caused by a mutation in either the TSC1 or TSC2 gene that encodes for the protein Hamartin and Tuberin respectively. The severity and phenotype of the disorder is incredibly varied and ranges from small skin fibromas to large brain tumours that can cause epilepsy and developmental delays. More severe manifestations of the disorder are commonly associated with mutations in the TSC2 gene. A primary canonical function for the protein Tuberin is to act as a GTPase activating protein to inactivate Rheb (Ras-Homolog Enriched in Brain) to control protein synthesis and inhibit a master regulator of protein translation, the mammalian Target of Rapamycin. Our lab has identified a unique function for Tuberin at the G2/M checkpoint of the cell cycle. In this capacity, Tuberin binds to the G2 Cyclin, Cyclin B1, and regulates the subcellular localization of this important regulator of cell division. This unique interaction provides new insight into cell cycle dynamics as it is possibly a switch for allowing cells to know when to enter mitosis. This thesis examines the cell biology effects of specific alterations to the Tuberin protein, several with relevance to human disease. This study has shown that post-translational modification of Tuberin by the kinases ERK1/2 abrogates binding between Tuberin and Cyclin B1 and increases frequency of mitotic cells. We further show that elevated levels of a truncated form of Tuberin lacking the GTPase domain results in increased cell proliferation, reminiscent of that seen in patients with TSC. Many missense mutations found in the clinic have been shown to disrupt the Tuberin-Hamartin complex and result in TSC, however biochemical/molecular biology approaches to date have resulted in conflicting data regarding the molecular role of specific residues within these proteins. Collectively, our data and previously published data supports the critical importance of more clearly understanding the 3-dimensional structure of the Tuberin-Hamartin complex and how it is altered in the presence of other binding partners such as Cyclin B1. Using a published cryo-EM crystal structure of Tuberin-Hamartin and computational modeling in silico approaches we attempted to predict the structural importance of specific TSC2 mutations. After testing both mutations from the literature and choosing new mutations, we have identified that using this predicative computational modeling does not work to accurately predict the effect of binding on the Tuberous Sclerosis Complex. These data presented in this thesis could provide insights into the triggers for mitotic onset and development of more effective methods of complex formation using new in silico methods.

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