Investigating NF-κB ubiquitination: an in vitro study

QH301 Biology;QH345 Biochemistry;QR180 Immunology

Mitchell, Jennifer P. ; Carmody, Ruaidhri

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: NF-κB, transcription, ubiquitination, post-translational modification, gene expression, in vitro.;   

Others  :  http://theses.gla.ac.uk/74343/8/2019MitchellPhD.pdf

来源: University of Glasgow

【 摘 要 】

NF-κB is a transcription factor family that controls the expression of hundreds of biologically important genes, many of which have essential roles in the regulation of inflammation. When its activity is dysregulated, this can lead to the development of chronic inflammatory diseases such as rheumatoid arthritis. Therefore, it is critical that NF-κB is tightly controlled. The p50 subunit of NF-κB lacks a transactivation domain so when present as a homodimer, it acts as a transcriptional repressor in macrophages to limit the expression of pro-inflammatory cytokines and promote the resolution of inflammation. The stability of p50 homodimers is an important determinant of this repressor function and is controlled by ubiquitin-triggered degradation. Despite this, relatively little is known about the molecular mechanisms that target p50 for degradation, or the cellular components that mediate p50 ubiquitination. By identifying the components of the ubiquitin-proteasome system (UPS) that target p50 for degradation, and in particular, the identity of an E3 ligase for p50, we can intervene therapeutically to prevent ubiquitination and degradation from occurring, and regulate NF-κB activity in a gene-specific manner. In this thesis, the relationship between the known E3 ligases for the other NF-κB subunits and p50 was explored through a series of in vitro assays. SOCS1 was observed to promote the ubiquitination and degradation of p50, although this was in some capacity that is independent of both its E3 ligase activity and the proteasome. The role of a known site of ubiquitination of p50 was investigated using a mutant monocytic cell line and was found to have influence over the levels of the other subunits of NF-κB. Furthermore, transcriptomic analysis of two E3 ligase knock-out macrophage cell lines revealed that these selectively control the expression of NF-κB target genes in response to TLR activation. Collectively, the data presented in this thesis advances our understanding of the ubiquitination-controlled regulation of NF-κB transcriptional activity.