Fluorescent Imaging,Mass Spectrometric Quantification And Activity Evaluation Of Cysteine Cathepsins In Cells

Complex biological processes highly depend on the involvement of proteolytic enzymes.Cysteine cathepsins form a special family of acidic proteolytic enzymes with a common active site cysteine(Cys25).They are expressed in almost all living organisms,existing in cellular lysosomes.The different subtypes of cathepsins independently or synergistically parlticipate in a series of key physiological and pathological processes such as proteolysis,immune regulation and cancer metastasis.Methodological developments of cathepsins quantification and their enzymatic activity evaluation are very much desired,which are the first necessary step in cathepsin-associated oncological and immunological research.Early detection methods of cathepsins,which mainly based on gene knockout and western blot,did promoted studies on the functionality of cathepsins at a proteomic level.Two main strategies have been developed so far:synthetic substrate and activity-based probe.They are capable of reporting the activity level of functional cathepsins in a complex proteome and molecular events in real-time through fluorescence imaging.However,accuracy and sensitivity of these methods need to be further improved for absolute quantification and activity evaluation of different subtypes of cathepsins.New methods that are able to selectively target different subtype of cathepsins,quantify multiple subtypes and evaluate their activities are very much desired,they will be very helpful for understanding synergistic/antagonistic interactions between the subtypes and concentration-dependent activity in a particular physiological or pathological process.In this thesis,we established an activity-based probing strategy.Localization,quantification and activity evaluation of intracellular cathepsins and detection of pH in the lysosome’s internal environment as well could be realized using ICPMS and LCSM.We obtained more comprehensive and accurate information on cathepsins via selective recognition,visualization and quantification,paving a new avenue for understanding the related physiological mechanism of cathepsins.The main contents are described in four chapters.In Chapter 1,I introduced background knowledge of cathepsins including its structure,catalytic mechanism and main functions.General methods for cathepsin analysis and their advantages and disadvantages were summarized.Then I presented my research proposal.In Chapter 2,based on activity based protein profiling strategies,we designed a cathepsin probe alkynyl-Epoxy inhibitor(Epoxy-Lys-AK),which specifically binds to cathepsin active site Cys25.Furthermore,fluorescent probe Epoxy-Lys-BODIPY and click-chemistry-mediated elemental probe Epoxy-Lys-Eu were synthesized using green fluorescent tag BOD IP Y and element tag N3-DOTA-Eu as reporter groups,respectively.A cathepsins fluorescence imaging and ICPMS absolute quantitative analysis methodology was established.In Chapter 3,Epoxy-Lys-BODIPY was used as a probe for fluorescence imaging of cathepsins.Distribution of cathepsins in living cells was observed.In addition,based on activity-based element labeling strategy we developed previously,cathepsin-specific europium labeling(Epoxy-Lys-Eu)was achieved via click chemistry.It was successfully used for the determination of three cathepsin subtypes(cathepsin B,S,L)using HPLC-ICPMS with species-unspecific 153Eu-isotope dilution.Cathepsin activity was also evaluated via Eu-signal intensity thanks to that Epoxy-Lys-Eu is an activity-based probe.Moreover,the covalent conjugation between Epoxy-Lys-Eu and cathepsin is pH-dependent,allowing us to probe the pH in the lysosome microenvironment.Finally,this method was applied to quantify the expression and biological activity of cathepsin B,S and L in normal hepatocytes and hepatoma cells;and to evaluate the pH of lysosome in vivo.In Chapter 4,I summarized the research work during my MSc study.Perspectives of ICPMS-based biochemistry-mediated cathepsin analysis are briefly outlined.