Aggregation Behavior And Mechanism Of Bromodomain In ATAD2

The ATPase family AAA domain-containing protein 2(ATAD2)is an epigenetic regulatory factor,also known as an oncogenic transcription factor.As a co-activator of transcription factors such as E2F,MYC,estrogen and androgen receptors,ATAD2 promotes downstream gene expressions,and forms an amplification cycle that improves cell proliferation,invasion and migration.The overexpression of ATAD2 is associated with various cancers,such as liver cancer,lung cancer,stomach cancer,prostate cancer,endometrial cancer,triple negative breast cancer,et al.Besides,ATAD2 up-regulation is also associated with poor prognosis of patients.Nowadays,ATAD2 has been taken as a prognostic marker in clinic and a potential drug target for cancer treatment.ATAD2 is composed of 3 domains:2 ATPase domains and a bromodomain.The bromodomain can specifically recognize acetylated lysines at the terminals of histones,through which regulates chromatin covalent modification,chromosome remodeling and transcription.The ATAD2 bromodomain is a"reader" of chromatinthat recognizes various acetylation modifications.It usually performs as a monomer in the cell.However,ATAD2 also exists in the form of oligomers.It has been reported that bromodomain protein CBP/P300 can produce amyloid aggregation with abnormal acetylated histones,and the addition of bromodomain inhibitors can reduce the amyloid aggregation of Huntington protein.These findings suggest that bromodomains are related to the formation of amyloid aggregation.The relationship between the aggregation and the function of ATAD2 bromodomian are still unknown.Because of its functional importance in chromatin recognition,it is essential to understand the mechanism of the aggregation which may influence the regulation of acetylated chromatin and the development of bromodomain inhibitors.In this paper,the structural characteristics and dynamic motions of ATAD2 bromodomain in solution was studied.The results showed that the ATAD2 bromodomain was prone to polymerize in the solution.1.By using transmission electron microscopy(TEM),circular dichroism(CD)and fluorescence spectroscopy,we observed that ATAD2 bromodomain suffers an amyloid aggregation with the conformational change.The secondary structure of the protein was transformed from α-helix dominated to β-sheets dominated over time.2.In order to explore the aggregation mechanism of ATAD2 bromodomain,nuclear magnetic resonance(NMR)technology and fluorescence spectroscopy were used to further detect the aggregation process of ATAD2 bromodomain,and analyze the possible factors that may trigger or affect the amyloid aggregation was analysed,such as temperature,protein concentration,pH,ion strength,etc.Meanwhile,because there are three cysteines in ATAD2 bromodomain,which may lead to format intermolecular disulfide bonds,the influence of reductant dithiothreitol(DTT)on protein amyloid aggregation was also studied.It gave a result that DTT could not stop the protein aggregation,but different concentrations of DTT have great influences on protein structure and aggregation rate.In addition,by the mutations of cysteines,we found that removing the free cysteine does not prevent the aggregation,whereas the knockdown of the disulfide bond within the hydrophobic cavity of the structure effectively alleviate the amyloid aggregation.These results indicate a critical role of the intramolecular disulfide bond in the aggregation.3.We investigated the effect of acetylated histone H4K5ac on amyloid aggregation of ATAD2 bormodomain.It was shown that H4K5ac significantly reduced the rate of amyloid aggregation,implying the contribution of H4K5ac binding site to the aggregation.Therefore,we propose that the aggregation behavior of ATAD2 bromodomain is related to the functional activities of the hydrophobic cavity,and the aggregation of ATAD2 bromodomain may be functionally required in cells.These findings provide helpful information for the development of inhibitors of ATAD2 bromodomain.