Characterisation of atypical human GTPases: Elucidation of molecular functions and interactors

Kay, Laura (2016) Characterisation of atypical human GTPases: Elucidation of molecular functions and interactors. Doctoral thesis, Northumbria University.

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Abstract

Emerging evidence suggests that several atypical Rho GTPases participate in the development of serious human pathologies such as chronic lymphocytic leukaemia and neurodegeneration. However, these GTPases remain poorly characterised at the functional, structural and biochemical level. Furthermore, the interactors and signalling pathways associated with these enzymes remain obscure. This study aimed to elucidate the molecular functions and interactors of three highly atypical human GTPases for the first time: hMiro1, hMiro2 and RhoH.

Using PCR-based cloning and molecular biology techniques, full-length and truncated versions of these highly unusual human GTPases were successfully expressed, purified and characterised. Colorimetric assays were utilised to assess the GTPase hydrolytic activity of the human Miros in vitro, while PC3 cell migration assays and fluorescent microscopy allowed for an evaluation of Miro GTPase influence in live cancer cells. Biophysical characterisation of the Miros and RhoH was performed using dynamic light scattering (DLS) and circular dichroism (CD) thermal denaturation, while label-free proteomics allowed for the identification of several novel putative Miro interaction partners. Meanwhile, RhoH association with the death-associated kinase DRAK2 was assessed in vitro using anti-phosphoserine/phosphothreonine assays.

The results show, for the first time, that individual domains of the human Miros are capable of intrinsic hydrolytic activity against GTP. Biophysical assessment by DLS suggests the Miros exist monomerically and exhibit average thermal stability. Knockdown of the human Miros produced a significant reduction in cell proliferation, with knockdown phenotypes associated with mitochondrial perinuclear aggregation under fluorescent microscopy. 37 novel putative signalling interactors for the human Miros were identified by mass spectrometry. Meanwhile, assessment of RhoH-DRAK2 interaction showed a novel association between these proteins in vitro, indicating that DRAK2 directly phosphorylates RhoH.

The results improve current understanding of these important and unusual human GTPases and the molecular functions they engage in. The novel interactors identified may also prove clinically relevant in the future, particularly to the understanding and treatment of neurodegenerative disorders and chronic lymphocytic leukaemia.

Item Type: Thesis (Doctoral)
Subjects: C400 Genetics
Department: Faculties > Health and Life Sciences > Applied Sciences
University Services > Graduate School > Doctor of Philosophy
Depositing User: Becky Skoyles
Date Deposited: 11 Oct 2018 09:30
Last Modified: 26 Oct 2019 08:08
URI: http://nrl.northumbria.ac.uk/id/eprint/36249

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