Development Of Fluorescence Methods For Protein Post-translational Modification Enzyme Assay

Protein post-translational modification(PTMs)refers to the chemical modification of one or more amino acid residues on the protein after the m RNA has been translated into protein.PTMs are key mechanisms to increase proteomic diversity.PTM enzymes are responsible for over 200 kinds of modifications of protein substrates and can be divided into two distinct mechanistic categories:(1)enzymes that hydrolyze peptide bonds(proteases)and(2)enzymes that covalently modify amino acid side chains.These enzymes can regulate protein activity,localization and interaction with other cellular molecules.Abnormal PTM enzyme activity is closely related to a variety of diseases,including diabetes,cancer,nervous system diseases,cardiovascular diseases,Alzheimer's disease,and so on.Thus,detection of PTM enzymes,is critical in the study of cell functioning and development of diagnostic and therapeutic tools.The conventional methods for the detection of PTM enzymes include radiometric assay,high-performance liquid chromatography(HPLC),gel-electrophoresis assay and enzyme-linked immunosorbent assay(ELISA).However,these methods involve radioactive elements,complicated sample preparation steps and expensive instruments.Therefore,we still need to develop simple,rapid and sensitive PTM enzyme detection methods to provide a powerful tool for clinical diagnosis and drug development of major human diseases.In this thesis,we use sortase A(StrA),matrix metalloproteinases(MMPs),protein tyrosine phosphatase 1B(PTP1B),protein kinase A as the models to develop a series of fluorescent methods for simultaneous detection of various PMT enzymes.These methods combinate with isothermal amplification technology,single-molecule detection technology,quantum dots(QDs)and magnetic beads,which have high sensitivity and specificity.The detailed contents are as follows:(1)The sortase-catalyzed coupling reaction is an efficient strategy to incorporate chemically defined modifications into proteins of interest.Despite its widespread applications in protein chemistry,the conventional bulk fluorescence measurement is not sufficient to characterize sortase due to the fluorescence inner filter effect-induced self-quenching.Herein,we develop a new method to visualize and quantify SrtA activity at the single-molecule level by using transpeptidation-directed intramolecular Fo?rster resonance energy transfer(FRET).This assay utilizes two cyanine dye-peptide conjugates,in which one carries an LPXTG motif and a donor fluorophore while the other harbors an oligoglycine nucleophile and an acceptor fluorophore as the substrate of SrtA.The presence of SrtA catalyzes the fusion of two conjugates and allows for the occurrence of intramolecular FRET.The FRET signal is recorded at the single-molecule level via total internal reflection fluorescence(TIRF)-based imaging.The proposed assay not only can accurately determine the kinetic parameters of SrtA but also can characterize the inhibition of SrtA activity by berberine chloride both in vitro and in Staphylococcus aureus cells.Moreover,the assay is very specific,and it can sensitively measure SrtA down to 7.08 pM,which is much lower than most of the reported methods.This strategy may provide a valuable tool for an in-depth study of sortases and for the discovery of anti-infective agents.(2)Protease expression is closely linked to various pathological phenomena,and their accurate quantification is essential to clinical diagnosis and cancer therapy.Herein,we demonstrate for the first time the construction of a sensitive protease sensor by integrating protease-sensitive cleavage with nicking enzyme-assisted signal amplification(NESA)for single-molecule detection of multiple matrix metalloproteinases(MMPs).This protease sensor involves two DNA-peptide conjugates which contain both specific protease cleavage sites and trigger DNAs and two report DNAs which are modified with a fluorophore(Cy3 or Cy5)and a quencher(BHQ2).In the presence of specific MMPs,MMPs-mediated cleavage reactions lead to the release of specific trigger DNAs from the corresponding DNA-peptide conjugates.After the magnetic separation,the resultant trigger DNAs may hybridize with the corresponding report DNAs to initiate the cyclic NESA reaction,releasing large amounts of Cy3/Cy5fluorescent molecules which can be simply quantified by using total internal reflection fluorescence-based single-molecule detection.Taking advantage of the high specificity of proteolytic cleavage,the high amplification efficiency of cyclic NESA,and the high sensitivity of single-molecule detection,this protease sensor can simultaneously detect multiple MMPs with a detection limit of 3.33 pM for MMP-2 and 1.71 pM for MMP-7,superior to the target peptide-based methods.Moreover,this protease sensor can be applied for the measurement of MMP-2 and MMP-7 in cancer cells and the screening of protease inhibitors,holding great promise in clinic diagnosis and drug discovery.(3)Tyrosine phosphorylation is a major regulatory mechanism for controlling various biological(e.g.,gene transcription,m RNA processing,and molecular transporting)and physiological(e.g.,proliferation,differentiation,and metabolism)activities.The intracellular level of tyrosine phosphorylation is rigorously regulated by a balanced interplay between protein tyrosine kinases(PTKs)and protein tyrosine phosphatases(PTPs).However,PTPs have always been undervalued in maintaining tyrosine phosphorylation status.Recent research studies indicate that protein tyrosine phosphatase 1B(PTP1B)may function as a key negative regulator in insulin/leptinmediated signaling,and its aberrant activity may contribute to type II diabetes and obesity.Moreover,the dysregulation of many other PTPs has been linked to the progression of various human diseases and cancers.Thus,the effective and sensitive detection of PTP activity is of great significance for clinical diagnosis,drug discovery,and cancer therapy.Conventional methods for PTP assay mainly rely on the measurement of the released radioactive phosphates from ³²P/³³P-labeled phosphorylated peptide/protein substrates,and the quantification of liberated radioactivity by liquid scintillation spectrometers.However,radioactive elements can cause hazards to human health and environments.We develop a new fluorescence method for the sensitive detection of protein tyrosine phosphatase 1B(PTP1B)based on dephosphorylation-directed tricyclic DNA amplification cascades.This strategy involves three steps:(1)PTP1B-catalyzed tyrosine dephosphorylation induces the cleavage of the peptide–DNA substrate by chymotrypsin,(2)peptide cleavage-directed exponential strand displacement amplification(SDA)produces DNAzymes,and(3)DNAzyme-mediated recycling cleavage of signal probes generates an enhanced fluorescence signal.Taking advantage of the high specificity of PTP1B-induced tyrosine dephosphorylation and chymotrypsin-mediated peptide cleavage,the high efficiency of DNAzyme-mediated tricyclic DNA amplification,and the low background signal resulting from magnetic separation,this method exhibits good specificity and high sensitivity with a detection limit of 0.24 pM.Moreover,it can be applied for kinetics analysis,inhibitor screening,and the accurate detection of PTP1B in a variety of cancer cells.(4)Kinases can catalyze the transferring of?-phosphate group from adenosine-5'-triphosphate(ATP)to the recognition sites of a given target and plays a crucial role in various biological processes,including signal transduction,metabolism,gene expression,and cell-cycle progression.Protein kinases(PKA)and polynucleotide kinases(PNK)are two widely studied Kinases.Although great progress has been made in the detection of kinase,few universal kinases nanosensors have been reported so far.Herein,we demonstrate the zirconium ion-mediated assembly of a single quantum dots(QD)-based universal kinases nanosensor for the detection of both PKA and PNK.This nanosensor is constructed by the assembly of biotinylated capture probes onto the surface of single QD.In the presence of PKA and PNK,they can induce the phosphorylation of Cy5-modified peptide/DNA substrates,which can assemble onto the surface of single QD via the unique coordinative interactions between zirconium ions(Zr⁴⁺)and phosphate,resulting in the occurrence of fluorescence resonance energy transfer(FRET)between the QD and Cy5.The FRET signal can be easily measured by single-molecule detection.The proposed nanosensor is very sensitive with a detection limit of as low as 8.82×10^-4 U/mL for PKA and 1.40×10^-5 U/mL for PNK.Moreover,this nanosensor can measure intracellular PKA and PNK in HeLa cells,and it can be used to analyze the enzyme kinetic parameters and screen the inhibitors of PKA and PNK,holding great potential for further application in drug discovery and clinical diagnosis.