Identification Of An Endogenous Ligand For Hypoxia-Inducible Factor 3α(HIF-3α) And Study Of Its Mechanism Of Action

Hypoxia-inducible factors(HIFs)are a group of transcription factors that modulate cellular responses to hypoxic conditions.Transcriptionally active HIFs are heterodimers each formed by a subunit and β subunit(also known as ARNT).There are three types of α subunits in mammals,namely HIF-1α,HIF-2α and HIF-3α,which are encoded by three separate genes.HIFs regulate signaling pathways involved in erythropoiesis,angiogenesis,and anaerobic metabolism,all of which are critical physiological functions in organisms.The activity of HIFs is largely dependent on the stability of the HIF-α subunits,which is regulated by the cellular oxygen concentration.Under normoxia,HIF-α subunits in cytoplasm are hydroxylated in an oxygen-dependent manner,and degraded by the ubiquitin-proteasome system.While under hypoxia,HIF-α subunits stay stable and translocate into nucleus,where they form heterodimers with ARNT to perform transcriptional regulation.HIF-α subunits all possess the ligand binding ability.Artificial chemicals have been shown to bind to and regulate HIF-1α and HIF-2α subunits.However,no endogenous molecules that directly bind to and regulate the activity of HIF-α subunits have ever been discovered.The discovery of endogenous ligands is critical for a comprehensive understanding of the regulation mechanism of HIFs signaling pathways.Compared with HIF-1α and HIF-2α,the structure and function of HIF-3α subunit have not been well studied.HIF-3α has multiple splice variants and was originally thought to be a negative regulator of HIF-la and HIF-2α.In recent years,it has been found that HIF-3α can also exert transcription factor activity to regulate the expression of lipid metabolism-related genes.And the expression level of HIF-3α is related to human obesity.However,the underlying molecular mechanisms need to be further studied.Screening and discovering the active endogenous ligands of HIF-3α would be of great significance for exploring the physiological function of HIF-3α.To explore the ligand-binding potential of HIF-3α,first we determined the crystal structure of HIF-3α-ARNT heterodimer.Through structural comparison and computational analysis,we found the volume of the HIF-3α PAS-B domain pocket was similar to that of HIF-2α PAS-B,which indicated that HIF-3α indeed has a potential ligand binding ability.By using the affinity selection-mass spectrometry(AS-MS)method to screen endogenous ligands targeting the three HIF-α subunits,we identified oleoylethanolamide(OEA)binding to HIF-3α.OEA is a member of the N-acylethanolamine family,a group of fatty acid derivatives,and has the functions of promoting anorexia and modulating lipid metabolism.Using protein thermal shift(PTS)assays for cross-checking,we confirmed the specificity of OEA binding with HIF-3α.And the binding affinity of OEA to HIF-3α was 14 μM,as measured by surface plasmon resonance(SPR).Subsequently,time-resolved fluorescence resonance energy transfer assay(TR-FRET)and fluorescence quantitative PCR assay showed that OEA was an agonist of HIF-3α,which can enhance the interaction between HIF-3α and ARNT,and promote the transcriptional activity of HIF-3α.To further explore the mechanism of OEA performing the agonistic function,we determined the structure of the HIF-3α-ARNT in complex with OEA,and found that binding of OEA stabilized the loop connecting ARNT PAS-A and PAS-B domains(A/B loop)at the heterodimer interface.The mechanism of OEA stabilizing the A/B loop was futher explored using molecular dynamics(MD)simulation and hydrogen-deuterium exchange mass spectrometry(HDX-MS).We found that binding of OEA stabilized multiple residues in Fa and Gβ regions of HIF-3α PAS-B,futher enhanced the interactions between the HIF-3α PAS-B and the A/B loop of ARNT.The hydrogen bonds between R303 of Gβ and A/B loop had the greatest effect on OEA promoting the stability of heterodimer.Next we extented our exploration on the agonistic mechanism to other HIF-α subunits.HDX-MS showed the HIF-2α agonist M1001 also promoted the stability of heterodimer through a similar mechanism,indicating the agonists stabilizing HIF-α heterodimers might share a common mechanism.OEA was not completely enclosed in the HIF-3α PAS-B pocket,and its ethanolamine group was partially exposed to solvent outside of the cavity.This binding mode is different from other ligands targeting HIFs,which may be the best model to study the ligand entry pathway of HIFs.Compared with the Apo structure,binding of OEA caused the loop-Aβ of the HIF-3αPAS-B domain to flip towards outside of the pocket.And the key role of loop-Aβ for OEA binding was verified by a series of biochemical and cell based experiments.Using MD simulations,we found loop-Aβ can flexibly open and close,switching between the Apo and ligand-binding conformations in solution state.And based on these results,we proposed the pathway of ligands entering the PAS-B pocket of HIF-3α.We further expanded the research object to other HIF-α subunits,and found HIF-2α may have a ligand entry pathway similar to HIF-3α through MD simulations and point mutation experiments.In conclusion,this study identified an endogenous metabolite OEA specifically bound to HIF-3α.Using multidisciplinary approaches covering structural biology,biochemistry,cell biology and computer simulations,we successfully elucidated the mechanism of OEA enhancing the stability of the HIF-3α-ARNT heterodimer and proposed a ligand entry route into the PAS-B pocket of HIF-3α,providing new strategies for the design of drugs targeting HIF-α.OEA is the first endogenous ligand discovered to directly bind to HIF-α and regulate its activity,indicating that HIFs can be regulated by endogenous metabolite molecules in addition to the oxygen concentration.Futhermore,as a compound capable of inducing anorexia and weight loss,OEA can serve as a molecular tool for exploring the physiological functions of HIF-3α in lipid metabolism.