Identifying interactions of a mitochondrial localized ligand activated nuclear receptor and mtDNA interacting proteins

• Published in: The FASEB journal 2020-04, Vol.34 (S1), p.1-1
• Main Authors: Waterman, Hannah, Keogh, Dwight, Bestwick, Megan L.
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2020.34.s1.07242

Abstract only Mitochondria are key cellular organelles due to their traditional role in cellular metabolism and energy production, but also for their role in other important signaling and biosynthetic pathways. Because of this it is expected there are many levels of regulation for monitoring and controlling mitochondrial biogenesis. Our aim is to determine mechanisms by which mitochondrial localized ligand activated nuclear receptors act as transcription factors, or other possible rolls, within the organelle by assessing interactions of proteins with mitochondrial DNA (mtDNA) and other mitochondrial proteins. The nuclear hormone receptor superfamily composed of the peroxisome proliferator‐activated receptors (PPARs) play a major regulatory role in basic and complex metabolic function as well as energy homeostasis. The peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand activated type II nuclear receptor that can translocate to the nucleus and the mitochondria. It is mainly expressed in adipose tissue and is a master regulator of adipogenesis through forming heterodimers with retinoid X receptors. PPARγ regulates fatty acid storage and glucose metabolism, has a role insulin sensitivity, and is responsible for leading to a number of diseases such as diabetes, obesity and atherosclerosis. We are specifically interested in characterizing the role PPARγ plays in the mitochondria and to the extent it is associated with the mtDNA transcription process. We have cloned h‐PPARγ in a pH6HTC vector which contains a His‐tag for recombinant expression in E. coli and purification via affinity chromatography. Our current work is to utilize the recombinantly expressed and purified PPARγ protein in sequence specific DNA binding assays, as well as utilize the purified protein in a human in vitro mitochondrial transcription assay using short DNA templates based on known binding sequences to assess transcriptional output. We are also conducting additional experiments using an mtDNA and protein cross‐linking with immunoprecipitation (mtDNA ChIP‐type) assay that assess specific DNA binding in purified mitochondria.