Development Of Methods For Cell Membrane Engineering And Sensing And Imaging Of The Related Enzyme Activities

Cell membrane is a dynamic barrier separating the cell from its surrounding environment.It is a complex interface composed of a variety of functional molecules such as proteins,lipids,and carbohydrates.Cell membrane plays an important role in regulating selective transport of molecules,cell-to-cell communication,and cell-environment interaction.What's more,cell surface composition determines all interactions of the cell with its environment.Therefore,engineering of cell membrane to control cell functions has attracted a lot of attentions.At present,common methods for cell membrane engineering include genetic manipulation,chemical modification,non-covalent modification,and enzyme-mediated modification,which have been widely used in the study of cell membrane function,intercellular interaction,and regulation of cell behavior.However,genetic manipulation and chemical modification inevitably interfere with the physiological functions of cells,and enzyme-mediated engineering technology currently existing is only applicable to cell membranes with special recognition sites,and is not universal.Non-covalent modification is simple for cell membrane engineering with low interference and high efficiency and universality,and has been widely used.However,due to the complex chemical coupling and purification process between the anchor group and the target,this kind of method is limited to anchoring small molecules such as nucleic acid and oligopeptide to the cell membrane,and active biological macromolecules such as proteins are not involved.Membrane protein is the main manifestation of membrane function,anchoring protein to the cell membrane can not only widen the design scope of sensors on the cell membrane,but also endow the target cell with new biological functions.The simple and rapid method of anchoring the active protein to the cell membrane has great application prospect in controlling the intercellular interaction to regulate the activity of the cell surface channel,realizing the targeted delivery of drugs,and even improving the effectiveness and safety of immune cell therapy.Therefore,simple methods for efficient anchoring the active protein onto the cell membrane with little interference are desirable.In this thesis,firstly,a truncated peptide derived from bee venom was studied.This natural peptide can insert into the artificial lipid membrane through electrostatic and hydrophobic interactions,and we named it membrane inserting peptide(MIP).Based on MIP,we developed a new method for anchoring the active protein on cell membrane,and applied it to in situ detection of protease activity on the cell membrane.Cell membrane proteases specificly cleave the protein or peptide substrates and enable them to perform their physiological functions,a process closely related to many human diseases,including cancer.Different to other classes of proteases,most membrane proteases can only keep their correct conformation and activity on the cell membrane.Therefore,it is extremely important to develop methods for in situ detection of membrane protease activity.However,due to the lack of the membrane engineering technique,the method of in situ detection of protease activity on membrane is still limited.The MIP-based method will provide an opportunity for in situ analysis of the function of protease on cell membrane.Lipid transferase-catalyzed proteins lipidation is a common post-translational modification process in eukaryotic cells.Protein lipidation mainly occurs on membrane proteins and membrane-related proteins,and directly participates in the regulation of physiological processes such as cell signal transduction and intracellular protein localization.Based on the hydrophobic interaction between the lipidied protein and the cell membrane,an enzyme-mediated membranes engineering method for anchoring the active proteins to cell membranes is developed.Accordingly,we developed a label-free and visual method for studying of lipid transferase activity,a key enzyme to protein lipidation.The main contents are as follows:(1)According to the property of the truncated melittin which can bind to artificial lipid membranes,we developed a peptide-based tool for membrane anchoring.Here,MIP was fused and labled with green fluorescent protein(GFP).The properties of MIP inserting to cell membrane and its potential to help active protein anchor to cell membrane were studied through fluorescence imaging.The results showed that MIP could effectively anchor GFP to the cell membrane within 15 min,and there was no metastasis or endocytosis within 2 h,indicating that MIP can quickly anchor the cell membrane with good stability.MTT results indicated the good biocompatibility of MIP.In addition,the states of MIP on the cell membrane was analyzed through combining MIP with the technique of bimolecular fluorescence protein complementation.When GFP1-10(the 1^st-10^th?-strands of GFP)fused with MIP(MIP-GFP1-10)was inserted into the cell membrane,it can recombinant with the GFP11 fragment on the cell surface to form a complete GFP and emit green fluorescence.This result indicates that MIP embeds the N-terminal in the phospholipid bilayer and exposes the C-terminal outside of the cell membrane,as well as not affect the function of the target protein.With the advantages of small molecular weight,high insertion efficiency,good stability,low toxicity and designability,MIP has the potential to be a universal tool to anchor protein on cell membrane and be applied to cell membrane related research fields.(2)A novel semisynthetic green fluorescent protein assembly-based FRET probe(s FPAP)was developed by combining MIP and semisynthetic green fluorescent protein(GFP),which enables in situ,real-time and sensitive detection of protease activity on the cell membrane at single-cell level.The s FPAP probe is composed a MIP fused GFP1-10 and a synthetic peptide(P),where the GFP11 strand(the 11th?-strand of GFP)is integrated with a protease substrate sequence and tetramethylrhodamine(TAMRA)together.It was demonstrated that GFP1-10 and P can self-assemble into a complete GFP,which forms a FRET pair with TAMRA,and the FRET efficiency was calculated as high as 77.5%.With the help of MIP,the probe successfully anchored on the cell membrane.When the enzyme specifically recognizes and cleaves the substrate peptide,TAMRA moves away from GFP and reduces the FRET efficiency.Therefore,the increase in the ratio of F_GFP/F_TAMRAcan dynamically monitor protease activity and further reflect the expression level of protease on the cell membrane.The activity of furin with the detection limit as low as 3.1×10^-7 U/?L was measured,with the linear range of concentration from 1×10^-6 to 1×10^-4 U/?L.Compared with other membrane protease probes,s FPAP avoids complicated modification and purification processes in vitro,and has the advantages of simple,good biocompatibility,and high sensitivity.Since peptide P is highly designable,the s FPAP probe shows great potential as a versatile platform for cell-surface proteolytic enzymes assays.(3)Based on the lipid transferase-catalyzed protein lipidation,a new enzyme-mediated cell membrane engineering method was developed.Here,Hs NMT1(Homo sapiens N-myristoyltransferase 1)was selected as a model,and its specific substrate polypeptide(Pep)was fused with GFP(Pep-GFP).With the catalysis of Hs NMT1,the lipid myristate(C14 saturated fatty acid)from myristoyl coenzyme A(C14-Co A)is covalently linked to the N-terminal glycine of Pep-GFP to form C14-Pep-GFP,and then the lipid protein anchored on the cell membrane though hydrophobic interaction.Compared with the MIP,this method can anchor the fluorescent protein on the cell membrane more quickly in 10 min with a concentration as low as 4?M.Moreover,the fluorescence around the cell membrane was almost not transfer among different cells within 2 h,which proved the excellent stability of this method.Additionally,the lipid product has no obvious toxicity to the cells after long-term incubation with the cells,indicating the good biocompatibility of the method.Futhermore,the method can anchor not only GFP but also other proteins(streptavidin,STV)on the cell membrane of different types of cells,which suggested the universality of the method.Utilizing the naturally enzyme-catalyzed process,this work provides a new strategy for anchoring the active proteins on the cell membrane with little interference,and has a great prospect in the field of membrane labeling and even membrane related diseases.(4)Combining the process of lipid transferase-catalyzed protein lipidation and the unique optical properties of gold nanoparticles(Au NPs),a dual-product synergistic enhancement colorimetric method was constructed to quickly and sensitively detect of lipid transferase activity.Here,Hs NMT1,one of the key lipid transferases,was selected as the model.Accordingly,positively charged substrate peptides(Pep)of Hs NMT1 can induce the aggregation of Au NPs through disrupting their electrostatic repulsion.The Hs NMT1-catalyzed myristoylation generates aggregated lipidated peptides(C14-Pep)and negatively charged HS-Co A,which reverses the charge of biomolecules in the solution,and it will eliminate the disruption,and stabilize the Au NPs by the formation of Au-S bonds,respectively,and synergistically contribute to the stability of Au NPs.Therefore,the Hs NMT1 activity can be visually detected by naked eyes through the color change of the Au NPs.The ratio of A₅₂₀/A₆₁₀ can sensitively reflect the activity of Hs NMT1 in the linear range of 2-75 n M,and the lowest detection limit is 0.56 n M.Moreover,the method was successfully applied for probing the Hs NMT1 activities in different cell lysates,as well as inhibitor screening.Furthermore,given the flexible designability of the substrate peptide,the proposed assay is promising for universal application to other lipid transferases and exhibits great potential in lipid transferase-targeted drug development.