Mechanism Research Of PRMT1 Promoting Tumor Progression Via MTORC1 Signaling Pathway

Backgroundm TORC1 is a master regulator of cell growth and homeostasis in response to diverse environmental cues,including amino acids^[1,2].Dysregulation of m TORC1 signaling has been linked to many human diseases,including cancer,diabetes and neurological disorder^[3–5].Amino acids signal is transmitted to m TORC1 primarily through the Rag GTPase via promoting the lysosome translocation of m TORC1 and its subsequent activation by the small GTPase,Rheb^[6–9].A plethora of protein complex have been identified to govern the amino acid sensing and subsequent activation of m TORC1 via regulating the activation status of Rag GTPases.Furthermore,Regulator controls lysosome localization of Rag GTPase and executes its GEF activity toward Rag C^[10–12].On the other hand,FLCN-FNIP and GATOR1have been identified to function as GAPs for Rag C/D and Rag A/B,respectively^[16,17].The KICSTOR complex tethers GATOR1 to the lysosome surface and is essential for amino acid starvation to suppress m TORC1 activity^[18,19].Further studies reveal that GATOR2 is capable of interacting with and potentially antagonizing GATOR1’s activity,which is required for the m TORC1 activation,but the detailed mechanism underlying GATOR2-mediated inactivation of GATOR1 remains enigmatic^[17].Recent studies have demonstrated that various amino acid sensors can dictate m TORC1signaling largely by interacting with the GATOR2 complex in an amino acid sensitive manner.Sestrin2,SARB1 and Castor1 directly bind to cytosolic leucine or arginine,integrates leucine and arginine availability to m TORC1 via GATOR2-GATOR1-Rag signaling axis^[20–22].Unlike leucine and arginine,methionine is sensed indirectly through the methionine cycle metabolite,SAM^[23].In the condition of methionine scarcity,the intracellular levels of SAM are dramatically reduced below the dissociation constant of SAMTOR with SAM,which promotes the interaction of SAMTOR towards GATOR1 and suppresses m TORC1 activation depending on SAMTOR^[23].However,though SAMTOR itself binds and senses methionine availability in an enzymatic independent manner^[23].Thus,it remains unknown mechanistically how the GAP activity of GATOR1 is suppressed upon methionine stimulation.Objectives1.To search for the regulator of GATOR1 and screen novel NPRL2 interacting proteins.2.To verify that PRMT1 can participate in m TORC1 activation under amino acid stimulation and further explore the molecular mechanism.3.To verify the role of PRMT1 in the activation process of m TORC1 and its effect on tumors by animal experiments and clinical specimens.Methods and Results1.Immunoprecipitation,mass spectrometry analysis,and GST-pulldown assay demonstrated that that PRMT1 interacted with NPRL2 in cells2.WB demonstrated that in cells lackingk PRMT1,the amino acid-induced m TORC1activation effect was inhibited.Through immunofluorescence confocal experiments,we found that knockout of PRMT1 also affects the lysosomal translocation of m TORC1 under methionine stimulation.After overexpression of the active structure of the Rag GTPase（Rag AQ66L/Rag C S75N）,or knockout of NPRL2,the deletion of PRMT1 no longer hinders the activation of m TORC1.3.We found through immunoprecipitation experiments that methionine stimulation can inhibit the interaction between SAMTOR and NPRL2 and strengthen the interaction between PRMT1 and GATOR1.Overexpression of SAMTOR,which can disrupt the interaction between GATOR1 and PRMT1 in a dose-dependent manner.After knockout of SAMTOR,the interaction ability of PRMT1 with GATOR1 was significantly improved and it was no longer dependent on concentration stimulation of methionine.After knockout of SAMTOR,the deficiency of PRMT1 no longer hinders methionine-mediated m TORC1activation.4.We found that knockout of PRMT1 inhibits methionine and SAM-mediated m TORC1activation.We used WB and immunofluorescence confocal experiments to find that the mutant forms of PRMT1（G98R and E162Q）with SAM binding ability deficiencies were unable to activate m TORC1 under methionine or SAM stimulation,as in cells knocked out of PRMT1.After we inhibited PRMT1 methyltransferase activity,the activity of methionine-induced m TORC1 could not be activated in the presence of GATOR1.5.We found that stimulation at methionine can lead to an increase in total asymmetric dimethylation（ADMA）modifications on lysosomal proteins.Through lysosomal immunoprecipitation experiments,we found that after knocking out NPRL2,PRMT1 is no longer localized on lysosomes.While knocking out SAMTOR,PRMT1 can still be localized on the surface of the lysosomal.6.Immunoprecipitation experiments demonstrated that methionine stimulation can modulate asymmetric dimethylation of NPRL2,a subunit of GATOR1,in a PRMT1-dependent manner.Inhibition or knockout of PRMT1 can downregulate asymmetric dimethylation levels of NPRL2.After mutating all five evolutionary conserved arginine residues of NPRL2 into lysine at the same time,PRMT1 could no longer promote its asymmetric dimethylation.Detection of lysosomal translocation of S6K phosphorylation and m TORC1,it was found that in NPRL2 methylation mimick mutant recombinant cells,methionine starvation or inhibition of PRMT1 could no longer inhibit the signaling of m TORC1.7.We purified the GATOR1 complex from HEK293T cells and performed an in vitro methylation assay,and we found that the unmethylated GATOR1 complex strongly stimulated GTP hydrolysis,while the methylation of PRMT1 significantly inhibited this hydrolysis process.We overexpressed and purified wild-type and methylated mimic mutants and found that methylated mimic mutants exhibited much lower GAP activity compared to wild-type GATOR1 complexes.8.By measuring the phosphorylation level of S6K,we observed a significant decrease in m TORC1 activity in fasting mice,while m TORC1 activity was enhanced after refeeding.Both the m TORC1 inhibitor rapamycin（10 mg/kg）or the PRMT1 inhibitor GSK3368715（100 mg/kg）can block the process of enhancing m TORC1 activity after refeed.After knocking out NPRL2 in hepatocytes,repeating the above starvation refeed,as well as GSK3368715 inhibition experiments,it can be observed that the inhibitory effect of the PRMT1 inhibitor GSK3368715 on m TORC1 signaling is no longer present.Therefore,we believe that PRMT1 can regulate the activation of m TORC1 in the liver through the subunit NPRL2 in the GATOR1 complex.9.Through metabolomics analysis,we found that the PRMT1 inhibitor GSK3368715 can block the synthesis of pyrimidine and purine precursor metabolites needed for cell growth.Through the detection of cell viability under methionine stimulation,we found that there was a significant decrease in cell viability when only PRMT1 was knocked out compared with the normal control group,while there was no significant difference between cell viability and normal control group when PRMT1 and NPRL2 were knocked out at the same time.10.In animal experiments,we injected human breast cancer cells subcutaneously into the abdomen of mice to form tumors.We observed that after inhibiting PRMT1,the mice had significantly smaller tumors than in the normal control group.Through the analysis of breast cancer bone metastatic tumor specimens collected in the clinic,we found that the signaling of m TORC1 was significantly reduced by inhibiting PRMT1 synthesis or inhibiting its enzyme activity.ConclusionsWe report that PRMT1 is required for methionine-mediated m TORC1 activation via directly sensing the intracellular levels of methyl donor SAM.In conditions of methionine sufficiency,elevated cytosolic SAM levels disassociate SAMTOR from GATOR1,which confers GATOR1 interaction with PRMT1 to methylate NPRL2 on multiple arginine sites to suppress its GAP activity,thus resulting in m TORC1 activation.Moreover,inhibition of PRMT1 impedes methionine-mediated m TORC1 activation in a GATOR1-dependent manner both in cells and in vivo.Our results therefore reveal PRMT1 as an enzymatic sensor of SAM,which connects methionine metabolism to m TORC1 activation.As inactivation of PRMT1 decreased tumor growth in a m TORC1-dependent manner,our results further implicate that pharmaceutical inhibition PRMT1 may provide novel therapeutic opportunities for the intervention of m TORC1 pathway in many cancers.