The topic of this thesis is the molecular regulation of Hypoxia-Inducible Factors (HIFs) by sumoylation. Hypoxia-Inducible Factors are transcriptional regulators that mediate cell survival during... Show moreThe topic of this thesis is the molecular regulation of Hypoxia-Inducible Factors (HIFs) by sumoylation. Hypoxia-Inducible Factors are transcriptional regulators that mediate cell survival during reduced oxygen conditions (hypoxia). HIFs are extensively controlled by post-translational modifications. The aim of this thesis was to obtain novel insight in the posttranslational modification of HIFs by sumoylation. Chapters 1 and 2 are introductions on sumoylation and Hypoxia-Inducible Factors, respectively. A summary of current knowledge is provided based on published literature on both systems. Chapter 3 describes the regulation of HIF-1_ transcriptional activity by sumoylation via a novel SUMO acceptor site. Chapter 4 describes crosstalk between sumoylation and ubiquitination, two similar but distinct post-translational modifications that act in concert to regulate the proteasomal degradation of a subset of protein targets. Chapter 5 shows that sumoylation of HIF-2_ marks it for proteasomal degradation during hypoxia, facilitated by E3 ubiquitin ligases RNF4 and VHL. Chapter 6 presents data on the formation of SUMO chains made up of a mix of SUMO-1 and SUMO-2/3 and we show that HIF-1_ is a target for SUMO chains in vitro. Chapter 7 provides a summary and overall discussion of the thesis. Show less
Hagen, M. van; Overmeer, R.M.; Abolvardi, S.S.; Vertegaal, A.C.O. 2010
Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1 alpha and... Show moreHypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1 alpha and HIF-2 alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2 alpha are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2 alpha. Functionally, sumoylation reduced the transcriptional activity of HIF-2 alpha. Similar to HIF-1 alpha, HIF-2 alpha is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2 alpha during hypoxia but did not affect the total level of HIF-2 alpha. The ubiquitin E3 ligases von Hippel-Lindau and RNF4 control the levels of sumoylated HIF-2 alpha, indicating that sumoylated HIF-2 alpha is degraded via SUMO-targeted ubiquitin ligases. Show less
