System Identification Of Small Molecular Directly Interacting Proteins And Mechanism Study Based On The Proteome Microarray

Small molecules(SMs)are ubiquitous in nature,and play important roles in biomedicine study and application,including two major categories: small molecular drugs,such as dihydroartemisinin(DHA),and biological small molecules,such as signaling molecule c-di-GMP.SMs carry out its function,such as curing diseases or regulating bioactivity,mainly through directly binding to proteins.The studying of SMs regulating function and clinical application relies on the knowledge of their targeting proteins,however for many SMs,the knowledge of the targeting proteins is still insufficient,especially in druggability and globality aspect.Traditional methods for identification of SMs targeting proteins,inculding affinity chromatography or Drug Affinity Responsive Target Stability(DARTS).Usually,these methods are unable to perform global identification,or difficult for direct targeting proteins identification.In order to better study SMs mechanism,it is necessary to develop a more systematic that enable direct targeting proteins identification.Proteome microarray is a high-throughput,direct,simple and fast tecnology for studying protein-protein,protein-nucleic acids,and protein-antibodies interaction.In addition,proteome microarray has also been applied in SMs targeting proteins identification,however,the procedure for this application has not been standardized yet.In our study,based on the proteome microarraytechnology,aimed at the second messenger c-di-GMP and dihydroartemisinin(DHA),which refer to the two diseases,i.e.,tuberculosis(TB)and systemic lupus erythematosus(SLE)respectively,we carried out the identification and mechanism studies of these two molecules,and established a standardization process for protein microarray based SM targeting proteins identification.Firstly,c-di-GMP is the procaryotic second messenger,and participates in numerous important physiological activities,such as biofilm formation,bacterial motility or virulence molecular regulation.However,there is unclear that what and how c-di-GMP perform function in mycobacteria.Tuberculosis(TB),caused by Mycobacterium tuberculosis(MTB),remains a threat to human health.To clarify the problem of c-di-GMP regulation in mycobacteria,we apply the MTB proteome microarray to identify 76 c-di-GMP directly binding proteins,including Eth R,a protein regulating ETH drug resistant.Combined a series of activity detection and drug-sensitive assay,we found that c-di-GMP enhanced the transcriptional repression activity of Eth R,in vitro and in vivo,and enabled M.sm to resist to anti-TB drug ETH.Then,we found that c-di-GMP could bind at Q125,R181,E191 sites in the central pocket of Eth R homodimer,which a highly conservative sites in mycobacteria.And we found that c-di-GMP could bind to parts proteins form Tet R family,suggesting the universality interaction between c-di-GMP and Eth R or Tet R family proteins.In order to investigate the possible new function of c-di-GMP regulation in mycobacteria,we performed the infection to THP-1 assay,stress assay,biofilm informationand etc.And we discovered that c-di-GMP could enhance the survival rate of M.sm in THP-1,affect survival rate of M.sm,colonial morphology and acid-fast staining under H2O2 stress,but do not affect survival rate of M.sm under NO stress and biofilm information.Thus,we identified a list of c-di-GMP binding proteins,and broadered the new function of c-di-GMP regulation in mycobacteria.Secondly,we have applied the human proteome microarray to systematic identify dihydroartemisinin(DHA)binding proteins.Except curing malaria,artemisinin and its derivatives,especially DHA,also has been found to be effective against other dieases,such as SLE,tumor,but the exactly mechanism is unclear.Firstly,we carried out the chemical synethsis,preparation of thin layer chromatography(PLC)and High Performance Liquid Chromatography(HPLC)purification,and Ultra Performance Liquid Chromatography-mass spectrometry(UPLC-MS)detection methods,we obtained the biotin-dihydroartemisinin(bio-DHA).Based on human proteome microarray,we identified 174 DHA binding proteins.With bioinformatics analysis,we have built a functional network of the DHA binding proteins,inculding glycometabolism,immune regulation,ubiquitin mediated proteolysis,chaperone and Ras signaling.Using western blot,we showed that 17 of 19 proteins of immune regulation,ubiquitin mediated proteolysis and chaperone pathway,bind to DHA,also suggesting the authenticity of the microarray results.Our finding may provides a valuable resource supporting for DHA relatedstudies,especially for curing SLE disease.Finally,by combining the above two studies of SMs’ targets discovery and our previous study on arsenic targeting proteins based on human proteome microarray,we standardized the process for identification of small molecule targets using proteome microarray.This standardized procedure is general,and it could be easily adopted for only SM of interest.Taken together,in this study,applying the MTB and human proteome microarray,we have systematically identified the targets and studied the related mechanism pathway of c-di-GMP and DHA.In addition,we have standardized the procedure of protein microarray based SM targeting proteins identification.Our discovery wides the function of c-di-GMP regulation and provides the support for curing TB.Meanwhile,the standardized procedure is generally applicable for other SMs.