Abstract
Recent work has shown that bilirubin has a hormonal function by binding to the peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor that drives the transcription of genes to control adiposity. Our previous in silico work predicted three potential amino acids that bilirubin may interact with by hydrogen bonding in the PPARα ligand-binding domain (LBD), which could be responsible for the ligand-induced function. To further reveal the amino acids that bilirubin interacts with in the PPARα LBD, we harnessed bilirubin’s known fluorescent properties when bound to proteins such as albumin. Our work here revealed that bilirubin interacts with threonine 283 (T283) and alanine 333 (A333) for ligand binding. Mutational analysis of T283 and A333 showed significantly reduced bilirubin binding, reductions of 11.4% and 17.0%, respectively. Fenofibrate competitive binding studies for the PPARα LBD showed that bilirubin and fenofibrate possibly interact with different amino acid residues. Furthermore, bilirubin showed no interaction with PPARγ. This is the first study to reveal the amino acids responsible for bilirubin binding in the ligand-binding pocket of PPARα. Our work offers new insight into the mechanistic actions of a well-known molecule, bilirubin, and new fronts into its mechanisms.
Highlights
Published: 17 May 2021The peroxisome proliferator-activated receptors (PPARs) are a class of nuclear receptors that have become major targets for addressing various metabolic disorders, including lipid dystrophies and diabetes [1]
We found that bilirubin bound to albumin autofluoresces in a dose–response relationship and show bilirubin bound to peroxisome proliferator-activated receptor-α (PPARα) autofluoresces in a similar manner
We present bilirubin’s binding capacity with specific amino acids in the PPARα ligand-binding domain (LBD) and show that this can be quantitated based on its autofluorescence
Summary
The peroxisome proliferator-activated receptors (PPARs) are a class of nuclear receptors that have become major targets for addressing various metabolic disorders, including lipid dystrophies and diabetes [1]. PPARα and PPARβ/δ induce genes to regulate lipid uptake and metabolism [1,2,3]. PPARγ, which is stimulated by thiazolidinediones (TZDs; rosiglitazone, pioglitazone) [4], induces transcription of genes involved in fat storage and lipogenesis [1,2,5]. These nuclear receptors play key roles in regulating cellular metabolism, which results in phenotypic changes such as improved glucose storage and reduced serum lipids. Clinical phenotypes associated with mildly elevated bilirubin levels are associated with reduced adiposity, suggesting an interaction between
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