The Mechanism Of SIRT1 Regulating GCN2 Protein Expression

Post-translational modification(PTM)as a main regulatory mechanism in all organisms has been received more and more extensive attention and research.Diversified PTMs significantly increase the structure and fuction of proteins.The PTMs known nowadays is about 300 kinds.PTMs initiate and control many biological process,thus it is very important for us to investigate PTMs for studying the cellular regulatory mechanism.GCN2(general control nonderepressible 2)is one of the important regulatory factors in mammals,also known as eIF2aK4(eukaryotic initiation factor 2a kinase 4),which is a conserved serine/threonine protein kinase in structure and function.When faced with amino acid deficiency and other stress,GCN2 is activited and phosphorylate EIF2? at Ser52(yeast and mammals)to regulate cellular response to stress.Up to now,GCN2 protein expression regulatory mechanism is rarely known.In our previous study,GCN2 ubiquitination degradation mechanism was firstly analysed.It showed that NEDD4L was the specific E3 ubiquitin ligase enzyme of GCN2 and it would mediate GCN2 ubiquitination.Interestingly,the ability of NEDD4L recognizing and degrading GCN2 was weak and it needed ?-arrestins as scaffold protein to increase the relationship with GCN2.Thus,it is of great importance for us to discover further why NEDD4L had weak ability to degrade GCN2 protein.There are 7 members in mammalian deacetylase Sirtuin family,SIRT1-SIRT7.Among them,SIRT1 is most widely researched.Human SIRT1 protein consists of 747 amino acids,which has a high conserved catalytic structural domain.SIRT1 is a NAD-dependent deaceytlase,which plays an important role in mammalian homeostasis,aging,autophagy and apoptosis process.Apart from the role in histone,SIRT1 is also found to deacetylate some non-histone proteins,such as p53,transcription factor NF-?B,forkhead family and Ku70.In our study,it was discovered that GCN2 had acetylation modification and deacetylase inhibitor Sirtinol increased the GCN2 acetylation level.Moreover,overexpression of SIRT1 downregulated GCN2 protein level,whereas transfection of SIRT1 deacetylase mutant SIRT1H363Y failed to decrease GCN2 protein level.Treatment of SIRT1 activator Reveratrol decreased the protein level of GCN2 and treatment of SIRT1 inhibitor Sirtinol upregulated the protein level of GCN2.The same result was also shown in human non-small-cell lung carcinoma cell A549,human cervical cancer cell Hela and human hepatoma carcinoma cell HepG2.Then it was speculated that SIRT1 would induce the degradation of GCN2.It showed that proteasome degradation system inhibitor MG 132 reversed the downregulation of GCN2 induced by SIRT1,which indicated that the degradation was relevant to proteasome degradation system.The proteasome degradation system process needed protein ubiquitination.When treated with Sirtinol,GCN2 ubiquitination was decreased,which indicated that GCN2 acetylation level would affect its ubiquitination.Morever,Sirtinol would increase the protein level of GCN2 downstream gene p-EIF2a and CHOP.Interestingly,it was also showed that when NEDD4L knocked down,it attenuated the degradation of GCN2 induced by SIRT1,which indicated that the degradation was mediated by NEDD4L.Moreover,Sirtinol could reverse the the degradation of GCN2 induced by NEDD4L and reduced the interaction between GCN2 and NEDD4L.GCN2 is a macromolecular EIF2? phosphorylase kinase with multiple domains,including kinase domains as well as other regulation domains.In our study,it was indicated that SIRT1 interacted with PK1 domain of GCN2,and when mutated PK1138 lysine site to arginine site,PK1 degradation induced by NEDD4L and SIRT1 was decreased.It was also found that SIRT1 negatively regulated GCN2 protein expression in many tumor tissues.Using the analysis of TCGA clinical tumor sample database,it was indicated that in Adrenocortical carcinoma,Skin Cutaneous Melanoma,Uterine Carcinosarcoma,Pheochromocytoma and Paraganglioma,GCN2 expression was high,whereas SIRT1 expression was low,which indicated that SIRT1 and GCN2 had significant negative correlation in tumor samples.Our study firstly showed the interaction between two important protein SIRT1 and GCN2,and clarified the the mechanism of GCN2 degradation induced by SIRT1,which provided new sight in GCN2 protein modification mechanism and regulation mechanism in tumors.