A New Binding Partner Of TRPM8 Channel,An E3 Ubiquitin Ligase TRIM4

TRPM8,which belongs to the TRPM subfamily,is a non-selective cation channel protein with six transmembrane domains that are capable of passing monovalent and divalent cations with a permeability of Ca²⁺>K⁺>Na⁺,but the penetration between these cations.However,the rate of difference is very small.The human TRPM8 gene is located on chromosome 2(2q37.1)and has 24 exons with a full length of 102.12 Kb.The mature TRPM8 m RNA encodes a protein containing 1104 amino acid residues with a molecular mass of about 130 k Da.According to research,TRPM8 is expressed in all the thermostatic vertebrates studied.It is widely found in various tissues such as prostate,breast,colon,lung,pancreas,brain and skin.TRPM8 is diverse in its pathophysiological function due to its widespread presence.TRPM8 is involved in the regulation of cold sensation,an important cold sensory receptor that activates under cold stimuli to sense cold.At the same time,TRPM8 is also involved in the regulation of pain sensation,because the cold signal transmission mediated by TRPM8 can also help to relieve pain,which is also an important way to protect the body from pain.In addition,in tumor cells,the expression of TRPM8 channel is abnormal,which affects tumor cell proliferation,differentiation,apoptosis,migration,invasion and metastasis,and cell death.Abnormally high expression or dysfunction of TRPM8 in melanoma,prostate cancer,pancreatic cancer,breast cancer,gastric cancer,oral squamous cell carcinoma,bladder cancer,osteosarcoma,and glioma,but it is known about the cause of TRPM8 expression/function abnormality less.Early studies showed that TRPM8 new interacting proteins TCAF1 and TCAF2 were discovered by GST-Pull down combined mass spectrometry.Further studies showed that TCAF1 and TCAF2 can regulate TRPM8 channel function,and the regulatory mechanism is different.At the same time,another research group passed immunoprecipitation.Combining mass spectrometry found that the candidate protein UBA1 of TRPM8 interacting protein can regulate TRPM8 function by adding UBA1 blocker;we and other research groups have shown that PIP2 can regulate TRPM8 activation and desensitization;it has been reported that TRPM8 channel activity or expression is reported.Regulation of hormones,growth factors,phospholipase A2(PLA2),and G proteins.However,the precise mechanism for regulating the expression or function of TRPM8 needs further exploration.Post-translational modification of a protein is an important step in the biological activity of a precursor protein by chemical modification.Post-translational modifications of various proteins,such as glycosylation,methylation,phosphorylation,ubiquitination,palmitoylation and acetylation of proteins,are common in organisms,among which ubiquitination modification is common.TRIM4 is a member of the TRIM(Tripartite motif 4)family,an E3 ubiquitin ligase.In the RIG-I signaling pathway,which produces antiviral innate immunity,TRIM4 actively regulates viral-induced IFN-b and downstream gene transcription.In addition,TRIM4 also regulates hydrogen peroxide(H2O2)-induced cell death.TRIM4 is differentially expressed in different tissues of the human body and is abundantly expressed in most cancer cell lines.Subcellular localization studies indicate that TRIM4 forms a unique plaque-like structure with transient interactions between cytoplasm and mitochondria.Expression of TRIM4 induces mitochondrial aggregation and elevated mitochondrial ROS levels in the presence of H2O2.H2O2 sensitized cells induce death,while knockdown reverses this effect,and TRIM4 plays an important role in regulating oxidative stress-induced cell death.However,it has not been reported that TRIM4 can regulate ion channel function and ubiquitination regulates TRPM8 function.The aim of this study was to explore the post-translational ubiquitination modification of TRPM8,and to discover the new interaction candidate protein of TRPM8,E3 ubiquitin ligase TRIM4,by GST-Pull down combined mass spectrometry.Using MCF7 and HEK293T,Hela three cell lines as a model,using co-immunoprecipitation experiments(Co-IP)and GST-Pull down experiments,further confirmed the interaction between TRPM8 and TRIM4.It was also found that TRIM4also interacts with other TRP family proteins,TRPP3.Functionally,TRPM8 and TRIM4 were transfected into HEK293T cells,and electrophysiological patch clamp experiments confirmed that E3 ubiquitin ligase TRIM4 could down-regulate TRPM8channel current.Further studies revealed that TRIM4 reduced TRPM8 expression on cell membrane.At the same time,ubiquitination analysis further proved that TRIM4 can promoted K63 polyubiquitination of TRPM8 and down-regulate the expression level of TRPM8 on the cell membrane.Based on the above studies,it was confirmed that E3ubiquitin ligase TRIM4 can ubiquitinate TRPM8,thereby reducing the expression of TRPM8 on the cell membrane and its channel activity.