Functions And Molecular Mechanisms Of E3 Ubiquitin Ligase TRIM13 In LPS-triggered Innate Immune Response

Innate immunity serves as the first guarding line against invading pathogens.Upon the recognition of pathogen-associated molecular patterns（PAMPs）or damage-associated molecular patterns（DAMPs）via pattern recognition receptors（PRRs）,immune cells can initiate certain signaling pathway and then produce pro-inflammatory cytokines as well as type I interferons,restraining the damage caused by pathogen accordingly.Toll-like receptors（TLRs）,one of the PRRs studied earliest and most extensively,play indispensable roles in defending the infection of pathogens and sustaining the body’s homeostasis by triggering innate immune response.Infection with gram-negative bacteria lays a huge burden on human health.TLR4 on the cell surface of innate immune cells,especially macrophages,promotes the release of relevant cytokines in a myeloid differentiation primary response gene 88（My D88）-dependent or My D88-independent manner,therefore activating the downstream molecules such as nuclear factor-κB（NF-κB）,mitogen-activated protein kinase（MAPK）and TANK binding kinase 1（TBK1）-Interferon regulatory factor 3（IRF3）,so as to inhibit the infection caused by this kind of bacteria.Once the TLR4 response is overactivated,however,the pro-inflammatory cytokines are prone to be released overwhelmingly and lead to the so-called“Inflammation Storm”,causing sepsis or other autoimmune diseases subsequently.Therefore,it is of great importance to investigate how the TLR4 response is finely tuned so as to remain activated at a moderate level.As a core part of post-translational modification,ubiquitination of proteins extensively regulates the innate immune response.Currently,numerous studies have indicated that E3ubiquitin ligases possess significant functions in regulating the onset,maintenance and resolution of inflammation by ubiquitinating the downstream molecules of TLR-associated pathway.But,the interaction between E3 ligases and their substrate proteins that are not directly embraced in the TLR-associated pathway has not been extensively investigated,which warrants further and more thorough investigations and are expected to shed light on tight regulation of innate immune response.Previously,we report that TRIM13（Tripartite motif-containing protein 13）,a typical RING-contained and Endoplasmic reticulum（ER）-resided E3 ligase,restrains pathogenic DNA-activated cyclic guanosine monophosphate–adenosine monophosphate（c GAMP）synthase（c GAS）-stimulator of interferon genes（STING）response through the interaction with STING,a core adaptor in antiviral response,and degradation of it.Meanwhile,we found that TRIM13 deficiency promoted the expression of inflammatory cytokines induced by LPS,but the mechanism of that is still unclear.In this study,we focus on the functions and molecular mechanisms of TRIM13 in regulating LPS-triggered immune response.First of all,we knocked down the expression of TRIM13 using Trim13-specific si RNAs,and found that the levels of IL-6 and IFN-βexpression were upregulated after stimulating the primary macrophages with LPS,which suggested that TRIM13 might play a negative role in the TLR4 response.Then,we stimulated the Trim13-deficient primary macrophages with LPS and found that the expression levels of IL-6,TNFαand IFN-βwere upregulated.In vivo experiments,we established the mouse infection model by injecting LPS intraperitoneally,and found that the damage and inflammatory cell infiltration levels of lungs,liver and kidneys significantly increased in the Trim13-deficient mice compared with Trim13 wildtype counterparts.At the same time,the serum levels of inflammatory cytokines in Trim13-deficient mice were obviously upregulated,indicating that TRIM13 regulated LPS-triggered inflammatory response in a negative manner.To elucidate the molecular mechanisms for TRIM13-mediated regulation of the LPS-triggered response,we examined the alteration of relevant signaling molecules in TLR pathway by using wildtype and Trim13-deficient primary macrophages stimulated with LPS by western blotting.Consequently,we found that the phosphorylation of p65,inhibitorαof nuclear factor-κB（IκBα）and mitogen-activated protein kinase（MAPK）was significantly enhanced in Trim13-deficient macrophages,while the activation of TBK1 and IRF3 did not show any significant changes during this process.Hence,we concluded that TRIM13participated in the LPS-triggered TLR4 signaling pathway.Next,we conducted Mass spectrometry（MS）,Co-immunoprecipitation（Co-IP）and confocal microscopy assays to identify the molecule which TRIM13 interacted with,and found stromal interaction molecule 1（STIM1）had a potential to bind with TRIM13.Moreover,we found that this kind of interaction between TRIM13 and STIM1 was susceptible to the treatment with LPS.Moreover,we constructed the eukaryotic expression vectors of the full-length and truncated fragments of both TRIM13 and STIM1 respectively and conducted Co-IP using these vectors,and confirmed that TRIM13 interacted with the cytoplasmic domain of STIM1 by the transmembrane domain of it.To further explore the underlying molecular mechanism,we examined the ubiquitination of STIM1 with or without TRIM13 deficiency,and found that STIM1 was only slightly ubiquitinated after LPS treatments in the TRIM13-knockout cells while STIM1was highly ubiquitinated in wild-type cells.Subsequently,we conducted ubiquitination assay in vitro using ubiquitin with only one lysine residue retained,and found that TRIM13promoted the K33-linked ubiquitination of STIM1.In view of the ER-associated degradation（ERAD）function of TRIM13 and the ER-localized feature of STIM1,we used the ERAD inhibitor and overexpressed TRIM13,and found that TRIM13 contributed to the ERAD of STIM1,which was probably ascribed to the K33-linked ubiquitination of it.Finally,considering that STIM1 mainly regulates store-operated calcium entry（SOCE）,we detected the intensity of SOCE in TRIM13-deficient primary macrophages using cytoplasmic calcium fluorescence probe,and found that TRIM13 deficiency resulted in a significant enhancement of calcium entry.In light of the essential role of Ca²⁺in protein synesis in ER,we investigated the relevant molecules in ER stress,and found that TRIM13deficiency augmented the expression and phosphorylation of unfolded protein response（UPR）-associated molecules.Next,by overexpression of TRIM13 and using the inhibitor of Ca²⁺release activated Ca²⁺（CRAC）channel,we found that both rescue of TRIM13expression and inhibition of SOCE in TRIM13-deficiency macrophages resulted in an alleviation of ER stress,which suggested that imbalanced Ca²⁺homeostasis caused by TRIM13 deficiency could be proposed as an important mechanism leading to ER stress and activation of UPR.Furthermore,activation of IRE1α-XBP1s pathway might serve as a potential machinery promoting the intensity of TLR4 response.In summary,our study elucidates the mechanisms by which TRIM13 negatively regulates the TLR4 pathway.Briefly,TRIM13 promotes the ERAD of STIM1 to maintain the homeostasis of calcium store under the resting state,while the interaction is released upon the challenge of LPS,which may lead to the elevated calcium entry and subsequent activation of UPR and therefore enhance the production of pro-inflammatory cytokines.Under the circumstance of TRIM13 deficiency,STIM1 undergoes an uncontrollable accumulation and impairs the homeostasis of calcium and triggers ER stress,leading to the excessive activation of TLR4 response.Here,we reveal the underlying mechanism for TRIM13-mediated negative regulation of TLR4 response,and our study extends the understanding of TRIM13 in regulating innate immune response as well as sheds light on a potential target for treating TLR4-associated disease.