stabilization, PPAR alpha co-repressor

The transcription co-repressors, silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR) have been observed to compete with transcriptional co-activators for binding to nuclear receptors (including PPARα) thus suppressing basal transcriptional activity (Nagy et al 1999, Xu et al 2002).  Binding motifs for the co-repressors and co-activators to PPARα have been observed to be conserved, however the co-repressor blocks the AF-2 helix from adopting the active conformation, as demonstrated in x-ray crystallography results presented in Xu et al (2002).  PPARα agonists and antagonists have been developed to selectively enhance co-activator or co-repressor binding, respectively (Xu et al 2001, Xu et al 2002).  Regarding the present key event, the prior binding of a PPARα antagonists such as GW6471 can stabilize the binding of the co-repressors to the PPARα signaling complex suppressing nuclear signaling and thus downstream transcription of PPARα-regulated genes.  Additionally, a natural human variant (V227A) in the hinge region of PPARα has been demonstrated to stabilize PPARα/N-CoR interactions resulting in inhibited transactivation of downstream genes in hepatic cells (Liu et al 2008) which can ultimately lead to measurable phenotypic effects of decreased blood triglycerides (Chan et al 2016).  The Liu et al (2008) study also demonstrated that the transactivation activity in the V227A variant could be restored when N-CoR was silenced.

Wilbanks et al. (2014) and Gust et al (2015) demonstrated inhibition of human PPARα nuclear signaling in in vitro nuclear signaling bioassays in response to 2,4-dinitrotoluene(2,4-DNT) and 2-amino-4,6-dinitrotoluene (2A-DNT), respectively.  However, it is unknown if this response was manifested through the co-repressor binding stabilization that was identified in Xu et al (2002).

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

In Xu et al (2002), X-ray crystallography was used to characterize the suppressed PPARα signaling complex (PPARα / GW6471 / SMRT) and was compared against the activated PPARα complex which included binding of PPARα with the agonist GW409544 and the co-activator, steroid receptor coactivator-1 (SRC-1).  Liu et al (2008) conducted in vitro investigations comparing human PPARα WT versus the V227A mutant in cell-based assays evaluated using quantitative real-time RT-PCR, competitive binding assays, immunofluorescence, glutathione-S-transferase (GST) pulldown, chromatin immunoprecipitation (ChIP).  In Chan et al (2006) triglycerides were measured directly from human blood serum samples from populations having the wild type PPARα gene compared to those having the V227A variant genotype.

The effects of 2,4-DNT and 2A-DNT on PPAR signaling was investigated using nuclear receptor reporter assays were conducted for (PPARalpha, PPARgamma, PPARsigma, and RXRalpha human cell-based assays, (Indigo Biosciences).

The fundamental mechanics for co-repressor antagonism of nuclear signaling by SMRT and N-CoR have been observed to be equivalent when comparing humans and yeast (Nagy et al 1999).  Therefore, the taxonomic applicability should be broad across eukaryotes.  However, the mechanistic perspective for SMRT, N-CoR and PPARα interactions described above was developed exclusively with the human PPARα system.  Also, the PPARα nuclear signaling in response to 2,4-DNT and 2A-DNT was established in a human in vitro system.

Chan, E., Tan, C.S., Deurenberg-Yap, M., Chia, K.S., Chew, S.K., Tai, E.S., 2006. The V227A polymorphism at the PPARα locus is associated with serum lipid concentrations and modulates the association between dietary polyunsaturated fatty acid intake and serum high density lipoprotein concentrations in Chinese women. Atherosclerosis 187, 309-315.

Gust KA, Nanduri B, Rawat A, Wilbanks MS, Ang CY, Johnson DR, Pendarvis K, Chen X, Quinn Jr. MJ, Johnson MS, Burgess SC, Perkins EJ. 2015. Systems Toxicology Identifies Mechanistic Impacts of 2-amino-4,6-dinitrotoluene (2A-DNT) Exposure in Northern Bobwhite. BMC Genomics. 16:587  doi:10.1186/s12864-015-1798-4.

Liu, M.H., Li, J., Shen, P., Husna, B., Tai, E.S., Yong, E.L., 2008. A natural polymorphism in peroxisome proliferator-activated receptor-alpha hinge region attenuates transcription due to defective release of nuclear receptor corepressor from chromatin. Mol. Endocrinol. 22, 1078-1092.

Nagy L, Kao H-Y, Love JD, Li C, Banayo E, Gooch JT, Krishna V, Chatterjee K, Evans RM, Schwabe JWR: Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev 1999, 13(24):3209-3216.

Wilbanks, M., Gust, K.A., Atwa, S., Sunesara, I., Johnson, D., Ang, C.Y., Meyer., S.A., and Perkins, E.J. 2014. Validation of a genomics-based hypothetical adverse outcome pathway: 2,4-dinitrotoluene perturbs PPAR signaling thus impairing energy metabolism and exercise endurance. Toxicological Sciences. 141(1):44-58.

Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL, Oplinger JA, Kliewer SA, Gampe RT, McKee DD et al: Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors. Proceedings of the National Academy of Sciences 2001, 98(24):13919-13924.

Xu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG, Cobb JE, McKee DD, Galardi CM, Plunket KD, Nolte RT et al: Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPAR[alpha]. Nature 2002, 415(6873):813-817.