Applications for Chemical Probes of Proteolytic Activity

Matthew Bogyo1, Amos Baruch2, Douglas A. Jeffery2, Doron Greenbaum3, Anna Borodovsky4, Huib Ovaa4, Benedikt Kessler4

1 Stanford University, Stanford, California, 2 Celera Genomics, South San Francisco, California, 3 University of California, San Francisco, California, 4 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.17
DOI:  10.1002/0471140864.ps2117s36
Online Posting Date:  September, 2004
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Abstract

Recent genome sequencing projects have identified new peptidases in multiple organisms, many with unknown functions, suggesting the need for new tools to study these enzymes. This unit outlines selection and use of small‐molecule and protein‐based probes to covalently modify peptidases in complex cellular environments. These activity‐based probes (ABPs) have been designed based on well characterized peptidase inhibitor scaffolds, but make use of new techniques to greatly enhance their utility for studying families of related peptidases. In particular, ABPs can be used to track activity of peptidases in crude cell extracts, intact cells, and in vivo, allowing rapid purification and identification of labeled targets. They can be used with libraries of small molecules to rapidly assess potency and selectivity of compounds in complex, physiologically relevant samples. Probe selection, probe tagging using reporters, labeling of recombinant targets, crude protein extracts, and peptidase targets in cell culture systems, affinity purification of targets, and inhibitor screening using affinity probes are outlined.

Keywords: affinity labels; activity based probes; cysteine peptidases; serine peptidases; DUBs; affinity purification; inhibitor competition; inhibitor selectivity

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Labeling/Tagging of Small Molecule Activity‐Based Reagents Using the Iodogen Method
  • Alternate Protocol 1: 125I Labeling of Small Amounts of a Protein‐Based Probe
  • Alternate Protocol 2: Tagging Small Molecules with Biotin or Fluorophore
  • Detection of Labeled Probes
  • Basic Protocol 2: Detection of Radiolabeled Probes by Autoradiography
  • Alternate Protocol 3: Detecting Fluorescently Labeled Probes
  • Alternate Protocol 4: Detecting Biotinylated Probes
  • Alternate Protocol 5: Detecting HA‐Tagged Protein‐Based Probes Using Anti‐HA Immunoblot
  • Basic Protocol 3: Optimizing in Vitro Labeling of Peptidases by Activity‐Based Probes
  • In Situ Labeling of Lysosomal Cysteine Peptidases with Activity‐Based Probes
  • Basic Protocol 4: Labeling of Lysosomal Cysteine Peptidases in Intact Cells Using 125I‐Labeled Activity‐Based Probes
  • Alternate Protocol 6: Labeling of Lysosomal Cysteine Peptidases in Intact Cells Using Fluorophore‐Labeled Activity‐Based Probe
  • Alternate Protocol 7: Labeling of Lysosomal Cysteine Peptidases in Intact Cells Using Biotinylated Activity‐Based Probes
  • Basic Protocol 5: Strategies for Identification of Active Peptidase by Immunoprecipitation
  • Alternate Protocol 8: Affinity Purification of Biotinylated Enzymes
  • Basic Protocol 6: Activity‐Based Probe Competition Assays to Characterize Small Molecule Peptidase Inhibitors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Labeling/Tagging of Small Molecule Activity‐Based Reagents Using the Iodogen Method

  Materials
  • Compound to be labeled
  • 50% (v/v) ethanol
  • Iodogen, Iodogen‐coated beads, or Iodogen‐coated microcentrifuge tubes (all available from Pierce)
  • Chloroform
  • 100 mCi/ml Na[125I] (3.7 GBq/mmol; Amersham Biosciences)
  • 50 mM sodium phosphate, pH 7.4 ( appendix 2E)
  • Acetonitrile
  • C18 SepPak light gel cartridges (Waters)
  • Column stand
  • 10‐ and 30‐ml Luer‐Lok disposable syringes
  • Lead pig
  • γ counter and tubes
CAUTION: This procedure involves handling a γ‐emitting isotope and must be done with extreme care and in an assigned location with proper radiation safety equipment. Also see appendix 2B. Be sure to use tips with filters in them to minimize contamination of the pipettor with volatile iodine. It is difficult to avoid this contamination, so it is desirable to have a dedicated set of pipettors for work with volatile radioactive iodine. Purification of the labeled probe also removes all volatile free iodine, so pipet tips without filters can be used with purified labeled probe.

Alternate Protocol 1: 125I Labeling of Small Amounts of a Protein‐Based Probe

  Materials
  • Sephadex G‐25 coarse resin (Amersham Biosciences)
  • 0.2 mg/ml bovine serum albumin (BSA) in 50 mM sodium phosphate buffer, pH 7.5 (see appendix 2E for buffer), 4°C
  • Protein to be labeled
  • 50 mM sodium phosphate buffer, pH 7.5 ( appendix 2E)
  • 1 M Na 2HPO 4
  • 1 M NaH 2PO 4
  • Iodogen‐coated microcentrifuge tubes (see protocol 1, step )
  • 100 mCi/ml Na[125I] (3.7 GBq/mmol; Amersham Biosciences)
  • 0.4 mg/ml tyrosine
  • 0.1 M NaI (unlabeled)
  • Tabletop centrifuge with swinging‐bucket rotor (e.g., IEC Clinical)
  • 1‐ml syringes
  • Glass wool
  • 10‐ml round‐bottom Falcon tubes
CAUTION: This procedure involves handling a γ‐emitting isotope and must be executed with extreme care and in a designated location with proper radiation safety equipment. Also see appendix 2B.

Alternate Protocol 2: Tagging Small Molecules with Biotin or Fluorophore

  Materials
  • Amine‐containing reagent
  • DMSO
  • Diisopropylethylamine (DIEA)
  • NHS ester‐containing tag (e.g., biotin or BODIPY fluorophore)
  • C18 column (unit 8.7)
  • 50% (v/v) acetonitrile in H 2O
  • Liquid nitrogen
  • 50‐ml polypropylene tubes
  • Wide‐gauge needle
  • Additional reagents and equipment for HPLC (unit 8.7)

Basic Protocol 2: Detection of Radiolabeled Probes by Autoradiography

  Materials
  • Sample: probe‐modified protein mixture of interest
  • Fixation solution (see recipe)
  • 50% (v/v) methanol
  • Whatman 3MM filter paper
  • PhosphorImager (Amersham Biosciences; optional)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and autoradiography (unit 10.11)

Alternate Protocol 3: Detecting Fluorescently Labeled Probes

  • High‐intensity flat‐bed fluorescent scanner (e.g., Typhoon; Molecular Dynamics)

Alternate Protocol 4: Detecting Biotinylated Probes

  Materials
  • Sample: biotin‐modified protein mixture of interest
  • Immunoblot transfer buffer (see recipe)
  • Immunoblot blocking solution (see recipe)
  • Vectastain Elite Kit (Vector Laboratories)
  • PBST: PBS ( appendix 2E) containing 0.1% (v/v) Tween 20
  • Chemiluminescent HRP substrate (e.g., ECL, Pierce)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and protein transfer to PVDF or nitrocellulose membranes (unit 10.7)

Alternate Protocol 5: Detecting HA‐Tagged Protein‐Based Probes Using Anti‐HA Immunoblot

  Materials
  • Solution of peptidase (purified or crude)
  • 10 mM stock of ABP in 100% DMSO
  • Specificity control (see Critical Parameters)
  • Tris hydrochloride and Tris base
  • EDTA
  • Triton X‐100
  • 1 M dithiothreitol (DTT)
  • 10 mM stock of specific inhibitor in 100% DMSO
  • 4× SDS‐PAGE sample buffer (unit 10.1)
  • Silanized microcentrifuge tubes (National Scientific; appendix 3E)
  • 80°C heating block or water bath
  • 96‐well polystyrene plates
  • Additional reagents and equipment for SDS‐PAGE analysis of tagged ABPs (see protocol 4 or Alternate Protocol protocol 53, protocol 64, or protocol 75)

Basic Protocol 3: Optimizing in Vitro Labeling of Peptidases by Activity‐Based Probes

  Materials
  • Cells
  • Serum‐containing medium appropriate for cells
  • Radiolabeled activity‐based probe (e.g., [125I]JPM‐OEt, a cell‐permeable probe)
  • Inhibitor: e.g., 10 mM E‐64d (Calbiochem) in 100% DMSO
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Cell lysis buffer (see recipe)
  • 12‐well tissue culture plate
  • Rubber policeman
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), detemination of protein concentration (unit 3.4), and detection of radiolabeled probes (see protocol 4)

Basic Protocol 4: Labeling of Lysosomal Cysteine Peptidases in Intact Cells Using 125I‐Labeled Activity‐Based Probes

  • Serum‐free medium appropriate for cells
  • Fluorescently labeled activity based probe (e.g., BODIPY‐DCG‐04, a cell‐permeable probe)
  • Additional reagents and equipment for detection of fluorescently labeled probes (see protocol 5)

Alternate Protocol 6: Labeling of Lysosomal Cysteine Peptidases in Intact Cells Using Fluorophore‐Labeled Activity‐Based Probe

  • Biotinylated activity‐based probe (e.g., biotin‐DCG‐04, a non‐cell‐permeable probe)
  • Additional reagents and equipment for detection of biotinylated probes (see protocol 6)

Alternate Protocol 7: Labeling of Lysosomal Cysteine Peptidases in Intact Cells Using Biotinylated Activity‐Based Probes

  Materials
  • Biotinylated enzyme
  • Specific antibody and control antibody
  • IP wash buffer (see recipe)
  • Protein A or protein G coupled to agarose beads (Amersham Biosciences)
  • 10% (w/v) SDS ( appendix 2E)
  • RIPA dilution buffer (see recipe)
  • RIPA buffer (see recipe)
  • End‐over‐end rotator, 4°C
  • 80°C water bath
  • Additional reagents and equipment for labeling enzymes (see protocol 8) and SDS‐PAGE (unit 10.1; also see protocol 4)

Basic Protocol 5: Strategies for Identification of Active Peptidase by Immunoprecipitation

  Materials
  • Biotinylated enzyme of interest
  • PBS ( appendix 2E)
  • Streptavidin‐agarose beads (Pierce)
  • PBS ( appendix 2E)/0.1% (w/v) SDS
  • PD‐10 desalting columns (Amersham Biosciences)
  • 15‐ml conical tubes (Falcon)
  • 80°C water bath
  • End‐over‐end rotator
  • Tabletop centrifuge
  • Additional reagents and equipment for labeling of peptidases (see protocol 8), desalting (unit 8.3), and SDS‐PAGE (unit 10.1 and protocol 4)

Alternate Protocol 8: Affinity Purification of Biotinylated Enzymes

  Materials
  • Source of target enzyme(s) (in solution or in live tissue culture cells)
  • Activity‐based probe with a fluorescent or radioactive tag (see protocol 1 or Alternate Protocol protocol 21 or protocol 32)
  • Small molecule inhibitors to be tested: as 0.1 mM stocks in DMSO
  • Dimethylsulfoxide (DMSO)
  • Graphing program (e.g., Microsoft Excel)
  • 96‐well plate
  • Multichannel pipettor capable of dispensing 1 to 20 µl
  • Additional reagents and equipment for peptidase labeling in vitro (see protocol 4) or in situ (see protocol 9), SDS‐PAGE (unit 10.1 and protocol 4), and autoradiography (unit 10.11)
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Figures

Videos

Literature Cited

   Bogyo, M., McMaster, J.S., Gaczynska, M., Tortorella, D., Goldberg, A.L., and Ploegh, H. 1997. Covalent modification of the active site threonine of proteasomal beta subunits and the Escherichia coli homolog HslV by a new class of inhibitors. Proc. Natl. Acad. Sci. U.S.A. 94:6629‐6634.
   Bogyo, M., Verhelst, S., Bellingard‐Dubouchaud, V., Toba, S., and Greenbaum, D. 2000. Selective targeting of lysosomal cysteine peptidases with radiolabeled electrophilic substrate analogs. Chem. Biol. 7:27‐38.
   Borodovsky, A., Kessler, B.M., Casagrande, R., Overkleeft, H.S., Wilkinson, K.D., and Ploegh, H.L. 2001. A novel active site‐directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20:5187‐5196.
   Borodovsky, A., Ovaa, H., Kolli, N., Gan‐Erdene, T., Wilkinson, K.D., Ploegh, H.L., and Kessler, B.M., 2002. Chemistry‐based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem. Biol. 9:1149‐1159.
   Fersht, A. 1985. Enzyme Structure and Mechanism, 2nd ed. W.H. Freeman, New York.
   Greenbaum, D.C., Medzihradszky, K.F., Burlingame, A., and Bogyo, M. 2000. Epoxide electrophiles as activity‐dependent cysteine peptidase profiling and discovery tools. Chem. Biol. 7:569‐581.
   Greenbaum, D.C., Baruch, A., Hayrapetian, L., Darula, Z., Burlingame, A., Medzihradszky, K.F., and Bogyo, M. 2002a. Chemical approaches for functionally probing the proteome. Mol. Cell. Proteomics 1:60‐68.
   Greenbaum, D.C., Baruch, A., Grainger, M., Bozdech, Z., Medzihradszky, K.F., Engel, J., DeRisi, J., Holder, A.A., and Bogyo, M. 2002b. A role for the peptidase falcipain 1 in host cell invasion by the human malaria parasite. Science 298:2002‐2006.
   Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual, 1st ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Kessler, B.M., Tortorella, D., Altun, M., Kisselev, A.F., Fiebiger, E., Hekking, B.G., Ploegh, H.L., and Overkleeft, H.S. 2001. Extended peptide‐based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta‐subunits. Chem. Biol. 8:913‐929.
   Kidd, D., Liu, Y., and Cravatt, B.F. 2001. Profiling serine hydrolase activities in complex proteomes. Biochemistry 40:4005‐4015.
   Krupa, J.C. and Mort, J.S. 2000. Optimization of detergents for the assay of cathepsins B, L, S, and K. Anal. Biochem. 283:99‐103.
   Liu, Y., Patricelli, M.P., and Cravatt, B.F. 1999. Activity‐based protein profiling: The serine hydrolases. Proc. Natl. Acad. Sci. U.S.A. 96:14694‐14699.
   Meng, L., Mohan, R., Kwok, B.H., Elofsson, M., Sin, N., and Crews, C.M. 1999. Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc. Natl. Acad. Sci. U.S.A. 96:10403‐10408.
   Mikolajczyk, J., Boatright, K.M., Stennicke, H.R., Nazif, T., Potempa, J., Bogyo, M., and Salvesen, G.S. 2003. Sequential autolytic processing activates the zymogen of Arg‐gingipain. J. Biol. Chem. 278:10458‐10464.
   Patricelli, M.P., Giang, D.K., Stamp, L.M., and Burbaum, J.J. 2001. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site‐directed probes. Proteomics 1:1067‐1071.
   Rappsilber, J. and Mann, M. 2002: What does it mean to identify a protein in proteomics? Trends Biochem. Sci. 27:74‐78.
   Schaschke, N., Assfalg‐Machleidt, I., Lassleben, T., Sommerhoff, C.P., Moroder, L., and Machleidt, W. 2000. Epoxysuccinyl peptide‐derived affinity labels for cathepsin B. FEBS Lett. 482:91‐96.
   Shi, G.P., Munger, J.S., Meara, J.P., Rich, D.H., and Chapman, H.A. 1992. Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine peptidase. J. Biol. Chem. 267:7258‐7262.
   Vugmeyster, Y., Borodovsky, A., Maurice, M.M., Maehr, R., Furman, M.H., and Ploegh, H.L. 2002. The ubiquitin‐proteasome pathway in thymocyte apoptosis: Caspase‐dependent processing of the deubiquitinating enzyme USP7 (HAUSP). Mol. Immunol. 39:431‐441.
   Wilchek, M. and Bayer, E.A. 1990. Introduction to avidin‐biotin technology. Methods Enzymol. 184:5‐13.
Key References
   Havlis, J., Thomas, H., Sebela, M., and Shevchenko, A. 2003. Fast‐response proteomics by accelerated in‐gel digestion of proteins. Anal. Chem. 75:1300‐1306.
  In‐gel digestion of protein bands.
   Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins silver‐stained polyacrylamide gels. Anal. Chem. 68:850‐858.
  Silver staining and MS sequencing.
   Rappsilber, J. and Mann, M., 2002. See above.
  MS sequencing.
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