Long-chain Fatty Acyl-CoA Fluorescent Sensor Based On FadR Protein And Live Cell Imaging

Lipid modification is a kind of ubiquitous post-translational modification of proteins.Since the unique physical and chemical properties of lipids conferring specific structures and functions to target proteins,it is important significance to study the structure and function analysis of protein translation lipid modification for multi-level cross signal transduction pathways.Researches have demonstrated that most of the sources of lipid modification are endogenously activated long-chain fatty acyl-CoA in mammals.However,the corresponding detection techniques for the important regulation of lipid modification reactions such as long-chain fatty acyl-CoA are extremely scarce.Therefore,it is extremely urgent to develop new detection technologies.Fluorescent proteins are currently widely used for labeling proteins in vivo.Since these fluorescent proteins are composed entirely of gene coding,they are also called genetically encoded fluorescent sensors.Due to the fluorescent protein sensor as a natural protein probe,genetically encoded fluorescent protein sensor have significant advantages in accurately localizing subcellular structures,eliminating human interference,and in vivo applications relative to chemical small molecule fluorescent probes.Therefore,fluorescent protein-based sensor imaging has become an analytical and rapid detection technology in the field of life sciences and medicine in recent years,and has been widely used in real-time observation of various physiological processes in living cells.Based on the above theory,the key and focus of this project is to develop rapid,spatially specific,real-time,dynamic biosensor for long-chain fatty acyl-CoA detection.Meanwhile,this biosensor is applied to the monitoring of long-chain fatty acyl-CoA in various tumor cells and normal cells,and it is hoped that the nuances of the long-chain fatty acyl-CoA concentration can be identified,and it can be achieved high temporal and spatial resolution imaging of long chain fatty acyl-CoA in single cell and animals.The detailed research contents are as follows:1.The condensation of fatty acid with coenzyme A was carried out by using PyBOP as a condensing agent,and 11 short,medium and long chain acyl-CoA were synthesized.A long-chain fatty acyl-CoA fluorescent protein sensor with strong fluorescence response and good ligand specificity was screened by exogenous addition of fatty acyl-CoA.2.The fluorescent protein(cpYFP)was inserted into a specific key position of long-chain acyl-CoA specific binding protein(FadR)by molecular cloning technology,and 63 fluorescent protein sensors were constructed by site-directed mutagenesis and fluorescent protein linker optimization.The in vitro characterization results showed that the two sensors of the fluorescent protein insertion site H91 and the linker are SAG/GT and R/R have nanomolar affinity and strong fluorescence change for long-chain acyl-CoA,named Acy1CoASer 1 and Acy1CoASer2.3.Acy1CoASer 1 and Acy1CoASer 2 were transfected into HEK293T cells by lipofection,respectively,for cell imaging.After treatment of HEK293T cells with long-chain acyl-CoA synthetase inhibitor N-ethylmaleimide,Acy1CoASer 1 increased fluorescence with time,and Acy1CoASer 2 decreased fluorescence with time.The results showed that the two types of sensors can detect the dynamic changes of long-chain fatty acyl-CoA in cells.4.The genetically-encoded AcylCoASer biosensor developed in this study has the ability to detect long-chain fatty acyl-CoA in tumor cells.The discovery of this new sensor will also provide an innovative tool for studying the regulation mechanism of long-chain lipid-modified proteins in tumor.