Expression, regulation and fuction of small ubiquitin-related modifier-1. Interaction with nuclear proteins in vivo

Abstract: The small ubiquitin-related modifier-1 (SUMO-1) is a 101-amino acid, ~12 kDa protein that is 18% identical to ubiquitin. Posttranslational covalent tagging of proteins with SUMO-1 (SUMOylation) serves many functions. For example, regulation of protein stability and modulation of transcriptional activation. However, very little work has been done on the regulation of SUMO-1 expression itself and functional distribution of SUMO-1 in vivo. In particular, the molecular interactions of SUMO-1 and its target proteins observed in in vitro systems needs to be confirmed in vivo when they are expressed at endogenous levels. The aim of this thesis is to investigate the expression, regulation and function of SUMO-1 with particular emphasis on its roles in protein-protein interaction in mouse ovary, brain and heart in association with cell death. A constitutive expression of SUMO-1 in all tissues examined in both female and male mice, is consistent with a fundamental role for SUMO-1 in normal tissue physiology. The differences observed in levels of SUMO-1 expression in the various tissues may reflect the functional SUMO-1 output needed by the tissues to exert the desired effects.Ubiquitin and ubiquitin-related proteins have been suggested to be involved in reproductive functions, such as follicular growth, oogenesis, pregnancy and spermatogenesis. We used various in vivo experimental approaches, including gonadotropins, pharmacological inhibitors and LH receptor knockout (LuRKO) mice to elucidate the hormonal regulation of ovarian SUMO-1 expression in mouse granulosa cells. Our study demonstrated that stimulation of LH receptor results in reduction of SUMO-1 expression in parallel with induction of progesterone receptor (PR) isoforms in periovulatory granulosa cells. Furthermore, SUMO-1 is not only co-expressed but also interacts with PR during ovulation in the mouse ovary. In addition, we also demonstrated that functional PR isoforms play an important role in the induction of ovulation and the inhibition of granulosa cell apoptosis. Although SUMO-1 modified PR, leading to decreased PR transcriptional activity in vitro, it remains to be determined if sumoylation of PR contribute to ovulation in vivo. Sumoylation of proteins is involved in many cellular processes, including apoptosis. Therefore, we investigated how the modulation of apoptosis affects SUMO-1 expression during granulosa cell differentiation. Our study suggests that a variety of potentially lethal stimuli converted to intracellular signals increase SUMO-1 protein expression in periovulatory granulosa cells but not in preovulatory granulosa cells in vitro. It is well established that hypoxia induces gene expression. One of the best identified and characterized responses of hypoxia is the activation of hypoxia-inducible factor-1 (HIF-1), a transcription factor that is central for the cellular adaptation to oxygen limitation as it is known to upregulate genes involved in angiogenesis. HIF-1 is a heterodimer comprising an O2-regulated alpha subunit (HIF-1alpha) and a constitutively expressed beta subunit. In order to better understand the cellular role of SUMO-1 expression in brain and heart in vivo, we demonstrated that hypoxia increased both SUMO-1 and HIF-1alpha expression in neurons and cardiomyocytes. In searching for a novel interactor of SUMO-1 protein, we discovered direct protein-protein interaction events between SUMO-1 and HIF-1alpha in response to hypoxia in adult mouse brain and heart.