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Abstract
ObjectiveTo signal, FGF19 and FGF21 require co-receptor βKlotho (KLB) to act in concert with FGF receptors, and yet there is appreciable variance in the C-terminal sequences of these two novel metabolic hormones where binding is believed to be primary. We seek to determine the functional consequences for these amino acid differences and determine whether such information can be used to design high potency antagonists and agonists. MethodsWe employed a functional in vitro assay to identify C-terminal protein fragments capable of fully blocking KLB-mediated FGF19 and 21 receptor signaling. The key residues in each hormone responsible for support full bioactivity were identified through peptide-based Ala-scanning. Chemical optimization of the peptides was employed to increase their antagonistic potency. An optimized sequence as a substituted part of a full length FGF21 was assessed for enhanced FGFR/KLB-mediated agonism using tissue culture and obese mice. ResultsC-terminal FGF19 and FGF21 peptides of relatively short length were observed to potently inhibit the activity of these two hormones, in vitro and in vivo. These FGFs of different sequence also demonstrated a striking conservation of structural determinants to maintain KLB binding. A single C-terminal amino acid in FGF19 was observed to modulate relative activity through FGFR1 and FGFR4. The substitution of native FGF21 C-terminal sequence with a peptide optimized for the highest antagonistic activity resulted in significantly enhanced FGF potency, as measured by in vitro signaling and improvements in metabolic outcomes in diet-induced obese mice. ConclusionsWe report here the ability of short C-terminal peptides to bind KLB and function as antagonists of FGF19 and 21 actions. These proteins maintain high conservation of sequence in those residues central to KLB binding. An FGF21 chimeric protein possessing an optimized C-terminal sequence proved to be a super-agonist in delivery of beneficial metabolic effects in obese mice.
Highlights
Fibroblast growth factors 19 and 21 (FGF19 and FGF21) structurally belong to the FGF superfamily
We chemically synthesized peptides of varying length derived from the Ctermini of FGF19 and FGF21 to analyze their relative ability to inhibit signaling of these endocrine FGFs
The emerging perspective is that conventional FGFs, and in particular FGF1 that signals through FGF receptors (FGFRs), can display potent endocrine biology [51e53]
Summary
Fibroblast growth factors 19 and 21 (FGF19 and FGF21) structurally belong to the FGF superfamily. In contrast to the classical FGFs that function in an autocrine/paracrine manner, these proteins have endocrine capabilities due to their low affinity for heparan-sulfate proteoglycans [1e4] They both require transmembrane b-Klotho (KLB) as a co-factor to facilitate signaling through various FGF receptors (FGFRs) [5e8] and display overlapping metabolic pharmacology in rodents [9e11]. Consecutive C-terminal shortening diminishes FGF21 interaction with KLB to eventually eliminate the receptor-complex engagement [15,16]. The importance of these findings is underscored by the report that Fibroblast Activation Protein (FAP) degrades both termini to inactivate FGF21, and as such has been implicated in the pathophysiology of metabolic disease [17e20]. The sequence identity in the C-terminal region of the two proteins is less than 40%, which is seemingly low for a common binding site
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