The Mechanism Of TRIM15 Regulating IFN-β Production

The innate immune system is the first line of defensive mechanisms that protect hosts against invading microbial pathogens,which also is the foundation of the adaptive immunity.The tripartite motif containing（TRIM）protein as host restrictive factor inhibits viral replication and is implicated in signaling pathways regulation.TRIM5αinhibits human immunodeficiency virus type 1（HIV-1）transduction by binding to and destabilizing the HIV capsidlattice before reverse transcription is completed.In recent years,TRIM15 has been confered direct antiviral activity and regulate signaling pathways.Knockdown of TRIM15 enhances assembly and release of MLV and HIV-1.TRIM15 up-regulates the production of type I interferon and must function upstream or at the level of MAVS in the RLR pathway.But TRIM15does not interact directly with RIG-1,MDA5 and MAVS.The molecular mechanism of TRIM15 regulating innate immunityis not yet clear.In our study,we investigatethe effect of TRIM15 on the regulation of IFN-βby immunoprecipitationandprotein mass spectrometry.The results are as follows:1.The effects of TRIM15 on inducing type I interferonPrevious studies have shown that knockdown of TRIM15significantly diminished type I interferon induction.Toverify the effect of over-expression of TRIM15 on inducing IFN-β.The results of luciferase activity and Real-time PCR show that TRIM15 significantly enhances the levels of IFN-βm RNA.In addition,over-expression of TRIM15 significantly enhances phosphorylation of IRF3,and significantly reduces the VSV titer.These results indicate thatover-expression of TRIM15 can promote IFN-βproduction.2.TRIM15 interacts with the host proteinTRIM15 positively regulates the production of IFN-β.To investigate the molecular mechanism of regulating IFN-β,293T cell line stably expressing TRIM15is obtained by lentiviral expressionsystem.Thehost protein 2-oxoglutarate dehydrogenas（OGDH）,which interactes with TRIM15,is captured by immunoprecipitation and protein mass spectrometry.In order to further validate the interaction between TRIM15 and OGDH.Theresults of immunoprecipitation indicate that TRIM15 interacts with the OGDH.Previous report say that OGDH located in mitochondria and TRIM15 located in cytoplasm.TRIM15 is isolated from the mitochondria.Immunofluorescence shows that TRIM15 and OGDH locate both on cytoplasm.The immunoprecipitation show that the PRY-SPRY domain and coiled helix domain are the key domains of TRIM15 interacting with OGDH.3.The function of OGDH on the signal pathway of TRIM15 regulating IFN-βIn order to determine the effect of the interaction between TRIM15 and OGDH on proteinexpression.Western blot analysis show that TRIM15 induced the degradation of OGDH.However over-expression of TRIM15 does not degrade exogenous OGDH.In order to investigate the key domain of TRIM15 degradating OGDH,different deletant of TRIM15 are transfected into 293T cells.Western blot analysis shows the RING domain is not the key domain degrading OGDH.Immunoprecipitationshow that TRIM15 develop ubiquitination,and the deletion of the RING domain will weaken the ubiquitination;And OGDH also occur ubiquitination.To investigate the effect of TRIM15 on OGDH ubiquitination modification,His-TRIM15,OGDH-Flag and HA-ubi weretransfected into 293T cells.The results of immunoprecipitation show that TRIM15 does not affect the degree of ubiquitination of OGDH.In order to further explore the degradation pathways of OGDH,cells are treated with proteasome inhibitor（MG132）,lysosomal inhibitor（NH₄CL）and autophagy inhibitor（3-MA）.The results show that NH₄CL treatment will restore the degradation degree of OGDH.This suggests that TRIM15degrade OGDH relying on lysosomal pathway.TRIM15 degrade OGDH relying on lysosomal pathway.So what role does OGDHplay in TRIM15-modulating interferon?First of all,we explore the effect of OGDH on the production of IFN-β.Real-time PCR and IFN-βreporter luciferase activity show that knockdown of OGDH up-regulates the production of IFN-β.Knockdown of OGDH significantly enhances phosphorylation of IRF3.Thus,knockdown of OGDH up-regulates the production of IFN-β.The above results indicate that TRIM15 up-regulated IFN-β,TRIM15interacted with OGDH and degraded OGDH relying on lysosomal pathway.However knockdown of OGDH up-regulated IFN-β.Thus,TRIM15 may up-regulate IFN-βdepending on degrading OGDH.