Activity-based protein profiling: the serine hydrolases.
PDF (226K)
Journal: 2000/January - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 10611275
Abstract:
With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
Open in
PUBMED | PMC | Google Scholar | Wikipedia
Relations:
Content
Citations
(202)
References
(36)
Drugs
(1)
Chemicals
(3)
Organisms
(2)
Processes
(2)
Anatomy
(1)
Affiliates
(1)
Similar articles
Articles by the same authors
Discussion board
Proc Natl Acad Sci U S A 96(26): 14694-14699
PMID: 10611275

Activity-based protein profiling: The serine hydrolases

The Skaggs Institute for Chemical Biology and Department of Cell Biology, the Scripps Research Institute, La Jolla, CA 92037
To whom reprint requests should be addressed at: The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail: ude.sppircs@ttavarc.
Communicated by Julius Rebek, Jr., the Scripps Research Institute, La Jolla, CA
Communicated by Julius Rebek, Jr., the Scripps Research Institute, La Jolla, CA
Received 1999 Sep 13; Accepted 1999 Oct 28.
Copyright © 1999, The National Academy of Sciences

Abstract

With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.

Abstract

Serine hydrolases play important roles in numerous developmental and tissue-specific events in vivo, including blood coagulation (1), inflammation (2), angiogenesis (3), neural plasticity (4), peptide hormone processing (5), and T-lymphocyte-mediated cytotoxicity (6). Additionally, several human diseases are associated with dysfunctions in serine proteases and/or their endogenous inhibitory proteins, including hemorrhagic disorders (7), emphysema (7), and cancer (8). The large number of mammalian serine hydrolases identified to date is both impressive and perplexing, with the endogenous functions of many members of this enzyme family remaining unknown. As ORFs encoding putative serine hydrolases continue to accumulate in public databases (9–11), the need for alternative experimental methods to study these enzymes is evident. One attractive approach for the analysis of serine hydrolase function would be to characterize these enzymes collectively, rather than individually. In particular, the majority of serine hydrolases are potently and irreversibly inhibited by fluorophosphonate/fluorophosphate (FP) derivatives like diisopropyl fluorophosphate (12, 13), whereas cysteine, aspartyl, and metallohydrolases are for the most part inert to such agents. Moreover, the reactivity of FPs with serine hydrolases requires that the enzymes be in a catalytically active state (12–14). Accordingly, we hypothesized that an FP linked to a small molecule reporter group might serve as a potent and selective probe for monitoring simultaneously the activities of multiple serine hydrolases. In this manner, serine hydrolases could be visualized on a systems level of analysis, greatly accelerating the assignment of potential functions and malfunctions to members of this enzyme family. Here, we report the synthesis and characterization of a biotinylated long-chain fluorophosphonate, referred to as FP-biotin and highlight its utility as an agent for profiling dynamics in serine hydrolase expression and function.

Acknowledgments

We thank Drs. R. Lerner, N. Gilula, P. Schimmel, C.-H. Wong, J. Kelly, S. Licht, and M. Bracey for critical reading of the manuscript and helpful discussions. Protein digestion and mass spectrometry analysis were done by Dr. J. Leszyk at the University of Massachusetts Medical School Core Laboratory for Protein Microsequencing and Mass Spectrometry (Shrewsbury, MA). This work was supported by grants from the Searle Scholars Program (to B.F.C.), the National Science Foundation (to M.P.P.), and the Skaggs Institute for Chemical Biology.

Acknowledgments

Abbreviations

APHacylpeptide hydrolase
FAAHfatty acid amide hydrolase
FPfluorophosphonate/fluorophosphate
MALDImatrix-assisted laser desorption ionization
STIsoybean trypsin inhibitor
equivequivalent
FTMSFourier transform MS
DHB2,5-dihydroxybenzoic acid
APHacylpeptide hydrolase
Abbreviations

References

  • 1. Kalafatis M, Egan J O, van't Veer C, Cawthern K M, Mann K G. Crit Rev Eukaryotic Gene Expression. 1997;7:241–280.[PubMed]
  • 2. Clark J D, Schievella A R, Nalefski E A, Lin L L. J Lipid Mediat Cell Signal. 1995;12:83–117.[PubMed]
  • 3. Mignatti P, Rifkin D B. Enzyme Protein. 1996;49:117–137.[PubMed]
  • 4. Yoshida S, Shiosaka S. Int J Mol Med. 1999;3:405–409.[PubMed]
  • 5. Seidah N G, Chretien M. Curr Opin Biotechnol. 1997;8:602–607.[PubMed]
  • 6. Smyth M J, O'Conner M D, Trapani J A. J Leukocyte Biol. 1996;60:555–562.[PubMed]
  • 7. Kato G J. Hum Mutat. 1999;13:87–98.[PubMed]
  • 8. Declerck Y A, Imren S, Montgomery A M, Mueller B M, Reisfeld R A, Laug W E. Adv Exp Med Biol. 1997;425:89–97.[PubMed]
  • 9. Davies B J, Pickard B S, Steel M, Morris R G, Lathe R. J Biol Chem. 1998;273:23004–23011.[PubMed]
  • 10. Ishikawa K, Nagase T, Nakajima D, Seki N, Ohira M, Miyajima N, Tanaka A, Kotani H, Nomura N, Ohara O. DNA Res. 1997;4:307–313.[PubMed]
  • 11. Nelson P S, Gan L, Ferguson C, Moss P, Gelinas R, Hood L, Wang K. Proc Natl Acad Sci USA. 1999;96:3114–3119.
  • 12. Creighton T E Proteins: Structure and Molecular Properties. 2nd Ed. New York: Freeman; 1993. [PubMed][Google Scholar]
  • 13. Walsh C T Enzymatic Reaction Mechanisms. New York: Freeman; 1979. [PubMed][Google Scholar]
  • 14. Pineiro-Sanchez M L, Goldstein L A, Dodt J, Howard L, Yeh Y, Chen W T. J Biol Chem. 1997;272:7595–7601.[PubMed]
  • 15. Chaurand P, Luetzenkirchen F, Spengler B. J Am Soc Mass Spectrom. 1999;10:91–103.[PubMed]
  • 16. Kobayashi K, Lin L-W, Yeadon J E, Klickstein L B, Smith J A. J Biol Chem. 1989;264:8892–8899.[PubMed]
  • 17. Giang D K, Cravatt B F. Proc Natl Acad Sci USA. 1997;94:2238–2242.
  • 18. Glynn P, Read D J, Guo R, Wylie S, Johnson M K. Biochem J. 1994;301:551–556.
  • 19. Kam C-M, Abuelyaman A S, Li Z, Hudig D, Powers J C. Bioconjugate Chem. 1993;4:560–567.[PubMed]
  • 20. Winkler U, Allison N J, Woodard S L, Gault R A, Ewoldt G R, Kam C M, Abuelyaman A, Powers J C. Mol Immunol. 1996;33:615–623.[PubMed]
  • 21. Patricelli M P, Lovato M A, Cravatt B F. Biochemistry. 1999;38:9804–9812.[PubMed]
  • 22. Keshavarz-Shokri A, Suntornwat O, Kitos P A. Anal Biochem. 1999;267:406–411.[PubMed]
  • 23. Carlsen P H J, Katsuki T, Martin V S, Sharpless K B. J Org Chem. 1981;46:3936–3938.[PubMed]
  • 24. Cravatt B F, Giang D K, Mayfield S P, Boger D L, Lerner R A, Gilula N B. Nature (London) 1996;384:83–87.[PubMed]
  • 25. Deutsch D G, Omeir R, Arreaza G, Salehani D, Prestwich G D, Huang Z, Howlett A. Biochem Pharmacol. 1997;53:255–260.[PubMed]
  • 26. Monsees T K, Schill W B, Miska W. Adv Exp Med Biol. 1997;424:111–123.[PubMed]
  • 27. MacDonald R J, Southard-Smith E M, Kroon E. J Biol Chem. 1996;271:13684–13690.[PubMed]
  • 28. Patricelli M P, Cravatt B F. Biochemistry. 1999;38:14125–14130.[PubMed]
  • 29. Scaloni A, Jones W M, Barra D, Pospischil M, Sassa S, Popowicz A, Manning L R, Schneewind O, Manning J M. J Biol Chem. 1992;267:3811–3818.[PubMed]
  • 30. Yoshimoto T, Tabiri J, Kabashima T, Inoue S, Ito K. J Biochem. 1995;117:654–660.[PubMed]
  • 31. Patricelli M P, Lashuel H A, Giang D K, Kelly J W, Cravatt B F. Biochemistry. 1998;37:15177–15187.[PubMed]
  • 32. Schriemer D C, Yalcin T, Li L. Anal Chem. 1998;70:1569–1575.[PubMed]
  • 33. Bei R, Paranavitana C, Milenic D, Kashmiri S V, Schlom J. J Clin Lab Anal. 1995;9:261–268.[PubMed]
  • 34. Karr J F, Kantor J A, Hand P H, Eggensperger D L, Schlom J. Cancer Res. 1995;55:2455–2462.[PubMed]
  • 35. Polascik T J, Oesterling J E, Partin A W. J Urol. 1999;162:293–306.[PubMed]
  • 36. Bogyo M, McMaster J S, Gaczynska M, Tortorella D, Goldberg A L, Ploegh H. Proc Natl Acad Sci USA. 1997;94:6629–6634.
  • 37. Wilkins M R, Williams K L, Appel R, Hochstrasser D F Proteome Research: New Frontiers in Functional Genomics. Berlin: Springer; 1997. [PubMed][Google Scholar]
  • 38. James P. Q Rev Biophys. 1997;30:279–331.[PubMed]
  • 39. Dove A. Nat Biotechnol. 1999;17:233–236.[PubMed]
  • 40. Anderson N L, Anderson N G. Electrophoresis. 1998;19:1853–1861.[PubMed]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.