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Title: Modulation of metabolic pathways through vitamin D receptor and retinoid x receptor : impact of components of sumoylation machinery
Author: Venkatesh, Jaganathan
ISNI:       0000 0004 5993 289X
Awarding Body: Ulster University
Current Institution: Ulster University
Date of Award: 2016
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The effects of Vitamin D are mediated through vitamin D receptor (VDR) in conjunction with its heterodimeric partner, retinoid X receptor (RXR), both members of the nuclear receptor superfamily. Determining the factors that modulate VDR functionality is crucial for understanding the pleiotropic nature of the biological responses to vitamin D. Sumoylation is a dynamic, reversible, post-translational modification, in which Small Ubiquitin like MOdifier (SUMO) protein is conjugated with target substrate via a lysine residue, a process enhanced by several protein families that harbour E3- Ligase activity such a ,Erotein Inhibitor of Activated STAT (PIAS) proteins. Sumoylation may be reversed by Sentrin ,Eroteases (SENP), which cleaves SUMO from the substrate through its isopeptidase activity. Our laboratory has previously reported VDR to be modified by SUMO-2, which is enhanced by PIAS4. SUMO-2 conjugates with VDR at Lysine 91, although this is a minor acceptor site with the major point(s) of conjugation yet to be determined. The RXR partner is also sumoylated at Lysines108 and 245 with this conjugation reversed by SENP1. This study was carried out to evaluate how PIAS and SENP as components within the SUMO pathway may serve as co-modulatory proteins of the VDR transcriptional response and also the impact of sumoylation within RXR-driven activity. In this context, the study primarily focused on NR regulation of metabolism related genes. Overexpression of PIAS4 in LNCap prostate cancer cells resulted in a significant inhibition of VDR target genes and this effect was reversed in the presence of a PIAS4 mutant variant (W356A) that is deficient in E3 ligase activity indicating that the overall effect of sumoylation is to repress the vitamin D response. In addition, we identified that SENP1 can serve to directly reverse the modification of VDR with SUMO (desumoylation) Overexpression of SENPl resulted in increased levels of VDR transactivation, with no such effects noted when using a SENPl mutant lacking isopeptidase activity suggesting SENPl coactivates VDR signaling through direct SUMO deconjugation of the receptor. Determining the specific amino acids that serve as SUMO acceptor sites within VDR through an approach involving site-directed mutagenesis followed by functional screening did not yield a positive identification, hence a modified approach utilising mass spectroscopy was designed. In assessing the role of sumoylation within RXR-directed signaling, the mutant form of this receptor (K108/K245R) that is totally deficient in sumoylation exhibits a significant increase in transcriptional function when assessed as a RXR-RXR homodimer and also in the context of the RAR-RXR heterodimer. We extended our study to also examine the LXR/RXR heterodimer, a major regulator of metabolic processes and noted that the overexpression of a RXR that is deficient in sumoylation resulted in a significantly diminished transcriptional response of the LXRRXR heterodimer. Taken together, this project establishes sumoylation pathway as a crucial regulatory element in modulating the transcriptional activities of the nuclear receptors, VDR and RXR.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available