Regulation Of Stress Granules Dynamics By Hedgehog Signaling Proteins SPOP And Sufu

BackgroundStress granules（SG）are cytoplasmic aggregates that are formed under cellular stress,concomitant with global translation inhibition.SG harbors RNA binding proteins,apoptosis/survival related mRNAs and translation initiation factors.SG formation promotes cell survival and is involved in many human pathogenesis.In mammals,there is a vital signaling pathway called Hedgehog（Hh）signaling pathway.It is important during embryonic development and its impairment or over activation can lead to many developmental disorders and tumors.Hh pathway is activated by Hh ligand binding with Ptch1 that ultimately converts GliFL to GliA and triggers the expression of Hh target genes.Gli transcription factors are regulated by SPOP and Sufu.SPOP targets Gli2/3 for proteasome-mediated protein degradation while Sufu protects Gli2/3 from degradation and negatively regulates Hh pathway.Previously it is reported that SG regulates some signaling pathways by recruiting specific proteins e.g.Raptor,RACK1 and TRAF2.Their recruitment into SG modulates stress response programs and protects cells from apoptosis,which drives us to investigate the potential interplay between SG and Hh signaling pathway.ObjectiveTo identify the functional association of Hh signaling pathway and stress granules at SPOP and Sufu level for the very first time.It will help to understand the physiological and pathological significance of the crosstalk between Hh signaling and SG.MethodsThe unbiased screening was conducted by two methods to find out the common regulators of SG and Hh signaling.In the bioinformatic analysis,an online database was used and for IF screen,Hh components were transfected into HeLa cells and their localization into SG were assessed by Immunofluorescence experiment.Chemical SG inducers（AS,MG132,TG,Sorbitol,H₂O₂,DTT）and heat stress was used to induce SG in HeLa cells.The interaction of SPOP and Sufu with SG proteins was investigated by Co-IP and GST-pull down assays.SG protein Caprin1 phase transition by SPOP was checked by triton extraction method.SPOP and Sufu mutation primers were designed by using online NEBaseChanger software and then proteins were cloned by following site-directed mutagenesis protocol.SPOP and Sufu different length fragment primers were designed by NEBaseChanger software and cloned by PCR.Sufu internal deletions primers were designed by NEBaseChanger and cloned by double PCR.ResultsIn the bioinformatic analysis,SG and Hh pathway proteomes reveal the overlap of six proteins that includes PSMD2,TNPO1,CDC73,HDAC6,TUBA1C and VCP.Results were verified by in-vitro IF-screen where Hh components were transfected to He La cells and their localization into SG was assessed.IF-screen confirms that SPOP fully localizes in SG while Sufu partially localizes in SG upon stress treatment.However,prostate cancer associated SPOP mutants are defective to localize in SG.SPOP and Sufu localization in SG is not cell specific or stress specific.Keen observation reveals that nuclear SPOP inhibits SG whereas cytoplasmic SPOP localize into SG by interacting with SG protein Caprin1.Apart from interacting with Caprin1,SPOP also regulates the phase transition of Caprin1 from a soluble form to an insoluble form.Sufu regulates SG dynamics in two ways.It either inhibits SG（in most of the case）or partially localize to SG probably by interacting with SG protein TTP.We find out that Sufu N-terminal fragment consisting of 110-268 amino acids is crucial for SG inhibition,however,Sufu C-terminal fragment from 269 to 484 amino acids potentially localize into SG and promote SG assembly.It might possible that Sufu N-terminal has dominant negative effect on C-terminal that’s why Sufu full length inhibits SG in the majority of cells.Interestingly,we observed that Gli2-binding ability of Sufu is important for SG inhibition and the disruption of this interaction leads to Sufu localization in SG without its inhibition.Moreover,SG inhibition mechanism of Sufu is dependent on its IDR,which is also required for Gli2/3-Sufu dissociation upon signaling activation.Sufu with IDR deletion preferentially localizes in SG.We suggest that SG inhibition mechanism of Sufu might be dependent on its IDR and stress induced conformational switch of Sufu protein.ConclusionIn conclusion,our studies provide direct evidence that SGs are regulated by Hh signaling proteins SPOP and Sufu for the first time.This essential association of SG and Hh pathway may open a new point to understand the physiological and pathological significance of crosstalk between Hh signaling and SG,and to uncover the pathogenesis of Hh-related and SG-related diseases.