Year: 2014
Type: dissertation
Abstract: The protein family GTP-ase of immunity associated protein (Gimap) is expressed in all vertebrates and angiosperm plants. One member of this family, Gimap3, is a pseudogene in humans but is expressed in mice, mainly in immune tissues and leukocytes. Together with members of the Bcl-2 family, Gimap3 and its paralogue, Gimap5, are needed for the maturation and survival of T cells as well as the maintenance of T cell homeostasis. However the mechanisms underlying this process are still unknown. Autophagy-related protein 5 (Atg5), a component of the autophagy degradation system, also plays a role in T cell maturation, especially in the negative and positive selection of T cells. Preliminary genetic studies suggested that the stability of Gimap3 is dependent on the expression of Atg5 but, whether they interact directly, remains to be seen. Furthermore Gimap3 is the first nuclear gene shown to modify the segregation of mitochondrial DNA (mtDNA) in hematopoietic tissues, although through an unknown mechanism. The morphological changes of mitochondria through fission and fusion are also connected to the maintenance and inheritance of mitochondria and possibly also to the segregation of the mitochondrial genome. Pathogenic mutant mtDNA variants in somatic tissues are shown to affect the segregation pattern, which is linked to the severity and the onset of mitochondrial disorders. Therefore, understanding the mechanism of mtDNA segregation is critical for understanding the development of mitochondrial disorders. In this thesis, a co-immunoprecipitation protocol was optimized to study the protein interactions of Gimap3 in order to elucidate how Gimap3 functions in maturation and development of T lymphocytes and also, by what mechanism it modifies the segregation of mtDNA. To achieve reliable results, an antibody precipitating Gimap3 specifically and efficiently, and a detergent with good solubilization capacity were chosen. The background contaminants in elution were reduced by differential centrifugation and stringent washes. Due to the high levels of background contamination, preliminary crosslinking experiments were done to further decrease the background with even more stringent washes. In mass spectrometry analysis, one protein, vesicle trafficking protein SEC22b (SEC22b), was identified to potentially interact with transmembrane domain of Gimap3. Atg5 was not found to interact with Gimap3 in the conditions tested. Further studies are needed to confirm these results and to optimize the co-immmunoprecipitation method for full-length Gimap3 in order to discover more protein interactions as well as interactions with its N-terminus.
Author: Pilvi Ruotsalainen
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2238861013