Identification of Functionally Relevant Lysine Residues That Modulate Human Farnesoid X Receptor Activation

Abstract

Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, proteinprotein association, and protein-DNA interaction were investigated. Results demonstrated that K122R, K210R, K339R, and K460R mutants of hFXR significantly impaired target gene [organic solute transporter a/b and bile salt export pump (BSEP)] promoter reporter activity in a ligand-dependent fashion. None of the four mutants affected the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/retinoid X receptor (RXR) binding affinity but enhanced the interaction of FXR with lysine methyltransferase Set7/9 by ∼21%. K460R decreased the FXR interaction with Set7/9 by ∼45% but had no significant effects on interaction with RXR. Electrophoretic mobility shift assays demonstrated that hFXR-K210R and -K339R reduced the protein-DNA (IR1 element at hBSEP promoter) binding affinity by ∼80 and ∼90%, respectively. Computational-based protein modeling studies were consistent with these results and provided further insights into the potential underlying mechanisms responsible for these results. In conclusion, four highly conserved lysine residues of hFXR, K122, K210, K339, and K460, have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).

Diversity-Oriented Synthesis of Azapeptides with Basic Amino Acid Residues: Aza-Lysine, Aza-Ornithine, and Aza-Arginine

“…3 For example, acylation and methylation of lysine residues of histone proteins alter chromatin structure and gene regulation. 1,4 Cell-penetrating peptides, 5,6 such as Tat and penetratin, are composed of multiple basic residues.…”

“…Basic amino acid residues, such as lysine and arginine, function in numerous biological processes including post-translational modifications, transport across membranes, and as enzymatic cleavage sites . For example, acylation and methylation of lysine residues of histone proteins alter chromatin structure and gene regulation. , Cell-penetrating peptides, , such as Tat and penetratin, are composed of multiple basic residues.…”

A comprehensive review on the pancreatic lipase inhibitory peptides: A future anti-obesity strategy

“…Our data also supported the view that this residue binds to aromatic side chains of PHE78, TYR115, PRO181, PHE216, and HIS264. Apart from pi stacking, the ε-amino group of lysine residues could also contribute to hydrogen bonding, which is also important in the catalysis of ligand contact that may be critical for substrate specificity [54]. This view is further supported by the finding that the LYS of the LAPSTIK peptide interacted with TYR115 via a hydrogen bond (Table 5Sa).…”

Targeting Farnesoid X Receptor in Tumor and the Tumor Microenvironment: Implication for Therapy

“…As described by Sun et al, replacing lysine with arginine at the following positions (K122, K210, K229, and K460) of FXR alters the expression of FXR targets including OSTα/β) and BSEP in a ligand-dependent manner. Furthermore, the mutation of K210R may affect FXR heterodimerization with RXR and reduce protein-to-DNA interaction at the hBSEP promoter [ 56 ].…”