The Mechanism And Function Of ODR4 Ufmylation Modification

As a ubiquitin-like modification,Ufmylation is involved in a variety of cellular biological processes.Ufmylation mainly occurs on the cytoplasmic side of the endoplasmic reticulum.The transmembrane protein ODR4(Odorant response abnormal protein-4)is also localized to the endoplasmic reticulum and interacts with UFM1 specific protease UFSP2,which mediates Ufmylation and de-Ufmylation.However,whether ODR4 can be modified by Ufmylation is unknown,and the biological function of ODR4 and the regulation of ODR4 modification by Ufmylation have not been reported.Therefore,this study is focused on the function of ODR4 and its regulation by Ufmylation modification in endoplasmic reticulum homeostasis.In this study,we found that ODR4 can interact with UFL1,the E3 ligase in the Ufmylation modification system.A number of proteins interacting with ODR4 including UFL1 were identified by immunoprecipitation followed by protein mass spectrometry analysis.By using the Ufmylation modification assays,we demonstrated that ODR4 can be modified by UFM1.In addition,Ufmylation modification affected ODR4 protein expression.In order to clarify whether the Ufmylation affects the stability of ODR4 protein,we used cycloheximide(CHX)to inhibit protein synthesis.In the absence of Ufmylation,the stability of ODR4 protein was enhanced and the degradation of ODR4 was inhibited.Furthermore,we found that ODR4 protein was degraded by autophagy-lysosomal pathway.However,we found that Ufmylation of ODR4 did not affect the interaction between ODR4 and UFSP2.So far,there are few studies on the biological function of ODR4.In order to study the regulation of ODR4 by Ufmylation,CRISPR-Cas9 technology was used to construct ODR4 knockdown stable cell lines.We found that knockdown of ODR4 inhibited cell proliferation.We performed RNA sequencing on A549 cells with knockdown of ODR4.By gene ontology(GO)data analysis,it was found that the expression levels of endoplasmic reticulum complex as cytological components(CC)were enhanced.Interestingly,we observed the endoplasmic reticulum swelling in ODR4 knockdown cells detected by immunofluorescence experiments,accompanied by up-regulation of endoplasmic reticulum stress related proteins such as heat shock protein family A(Glucose regulated protein 78;Bip)and DNA damage inducible transcript 3(CHOP),indicating that ODR4 plays a role in endoplasmic reticulum homeostasis.We treated ODR4-deficient cells and control cells with endoplasmic reticulum stress inducers tunicamycin(TM)and thapsigargin(Tg).The results showed that endoplasmic reticulum stress inhibited cell proliferation in ODR4 knockdown cells.In addition,Tg was used to treat cells overexpressing ODR4,and it was found that overexpression of ODR4 could alleviate the inhibitory effect of endoplasmic reticulum stress on cell proliferation.ODR4 not only affects the homeostasis of endoplasmic reticulum,but also seems to play a unique role in the regulation of de Ufmylation modification.Our experimental results show that the Ufmylation is enhanced in the absence of ODR4.In addition,we shown that ODR4 involves in Ufmylation modification by interacting with UFSP2.In summary,this study has demonstrated that ODR4 is a novel substrate for Ufmylation modification.Ufmylation affects the stability of ODR4 protein,which is degraded by autophagy-lysosomal pathway.ODR4 is involved in the regulation of endoplasmic reticulum homeostasis and the regulation of Ufmylation modification within ER.In further study,we should identify the ODR4 Ufmylation modification sites and investigate whether the Ufmylation affects the activity of ODR4 on endoplasmic reticulum homeostasis,which may provide insights for the understanding the functions of ODR4 in ER homeostasis and in Ufmylation modification.