Startseite Specific targeting of human caspases using designed ankyrin repeat proteins
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Specific targeting of human caspases using designed ankyrin repeat proteins

  • Andreas Flütsch , Thilo Schroeder , Jonas Barandun , Rafael Ackermann , Martin Bühlmann und Markus G. Grütter EMAIL logo
Veröffentlicht/Copyright: 5. September 2014
Biological Chemistry
Aus der Zeitschrift Biological Chemistry Band 395 Heft 10

Abstract

Caspases play important roles in cell death, differentiation, and proliferation. Due to their high homology, especially of the active site, specific targeting of a particular caspase using substrate analogues is very difficult. Although commercially available small molecules based on peptides are lacking high specificity due to overlapping cleavage motives between different caspases, they are often used as specific tools. We have selected designed ankyrin repeat proteins (DARPins) against human caspases 1–9 and identified high-affinity binders for the targeted caspases, except for caspase 4. Besides previously reported caspase-specific DARPins, we generated novel DARPins (D1.73, D5.15, D6.11, D8.1, D8.4, and D9.2) and confirmed specificity for caspases 1, 5, 6, and 8 using a subset of caspase family members. In addition, we solved the crystal structure of caspase 8 in complex with DARPin D8.4. This binder interacts with non-conserved residues on the large subunit, thereby explaining its specificity. Structural analysis of this and other previously published crystal structures of caspase/DARPin complexes depicts two general binding areas either involving active site forming loops or a surface area laterally at the large subunit of the enzyme. Both surface areas involve non-conserved surface residues of caspases.

Keywords: apoptosis; cell death; DARPins; specific caspase targeting; X-ray crystallography
Corresponding author: Markus G. Grütter, Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, e-mail:
aThese authors contributed equally to this work.bPresent address: University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0629, USA.cPresent address: Nextech Invest Ltd., Scheuchzerstrasse 35, 8006 Zurich, Switzerland.dPresent address: The Rockefeller University, Laboratory of Protein and Nucleic Acid Chemistry, 1230 York Avenue, New York, NY 10065, USA.

Acknowledgments

Financial support of this work was provided by the Swiss National Science Foundation grant 310030-122342 to M.G.G. We thank Beat Blattmann and Céline Stutz-Ducommun from the NCCR crystallization facility for crystal screening and the staff of the X06SA beamline at the Swiss Light Source of the Paul Scherrer Institute (PSI) for their support during data collection. Dr. Christopher Weinert is acknowledged for his calibration data of the size exclusion column.

References

Amstutz, P., Binz, H.K., Parizek, P., Stumpp, M.T., Kohl, A., Grütter, M.G., Forrer, P., and Plückthun, A. (2005). Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins. J. Biol. Chem. 280, 24715–24722.Suche in Google Scholar

Binz, H.K., Stumpp, M.T., Forrer, P., Amstutz, P., and Plückthun, A. (2003). Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol. Biol. 332, 489–503.Suche in Google Scholar

Binz, H.K., Amstutz, P., Kohl, A., Stumpp, M.T., Briand, C., Forrer, P., Grütter, M.G., and Plückthun, A. (2004). High-affinity binders selected from designed ankyrin repeat protein libraries. Nat. Biotechnol. 22, 575–582.Suche in Google Scholar

Blanchard, H., Kodandapani, L., Mittl, P.R., Marco, S.D., Krebs, J.F., Wu, J.C., Tomaselli, K.J., and Grütter, M.G. (1999). The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis. Structure 7, 1125–1133.Suche in Google Scholar

Boucher, D., Blais, V., and Denault, J.B. (2012). Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis. Proc. Natl. Acad. Sci. USA 109, 5669–5674.Suche in Google Scholar

Bravman, T., Bronner, V., Lavie, K., Notcovich, A., Papalia, G.A., and Myszka, D.G. (2006). Exploring “one-shot” kinetics and small molecule analysis using the ProteOn XPR36 array biosensor. Anal. Biochem. 358, 281–288.Suche in Google Scholar

Duarte, J.M., Srebniak, A., Schärer, M.A., and Capitani, G. (2012). Protein interface classification by evolutionary analysis. BMC Bioinform. 13, 334.Suche in Google Scholar

Favaloro, B., Allocati, N., Graziano, V., Di Ilio, C., and De Laurenzi, V. (2012). Role of apoptosis in disease. Aging 4, 330–349.Suche in Google Scholar

Flütsch, A., Ackermann, R., Schroeder, T., Lukarska, M., Hausammann, G.J., Weinert, C., Briand, C., and Grütter, M.G. (2014). Combined inhibition of caspase 3 and caspase 7 by two highly selective DARPins slows down cellular demise. Biochem. J. 461, 279–290.Suche in Google Scholar

Fuentes-Prior, P. and Salvesen, G.S. (2004). The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 384, 201–232.Suche in Google Scholar

Grütter, M.G. (2000). Caspases: key players in programmed cell death. Curr. Opin. Struct. Biol. 10, 649–655.Suche in Google Scholar

Hanes, J. and Plückthun, A. (1997). In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942.Suche in Google Scholar

Hardy, J.A., Lam, J., Nguyen, J.T., O’Brien, T., and Wells, J.A. (2004). Discovery of an allosteric site in the caspases. Proc. Natl. Acad. Sci. USA 101, 12461–12466.Suche in Google Scholar

Kabsch, W. (2010). Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144.Suche in Google Scholar

McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. (2007). Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674.Suche in Google Scholar

McStay, G.P., Salvesen, G.S., and Green, D.R. (2008). Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 15, 322–331.Suche in Google Scholar

Merz, T., Wetzel, S.K., Firbank, S., Plückthun, A., Grütter, M.G., and Mittl, P.R. (2008). Stabilizing ionic interactions in a full-consensus ankyrin repeat protein. J. Mol. Biol. 376, 232–240.Suche in Google Scholar

Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255.Suche in Google Scholar

Rohn, T.T. (2010). The role of caspases in Alzheimer’s disease; potential novel therapeutic opportunities. Apoptosis 15, 1403–1409.Suche in Google Scholar

Roschitzki-Voser, H., Schroeder, T., Lenherr, E.D., Frölich, F., Schweizer, A., Donepudi, M., Ganesan, R., Mittl, P.R., Baici, A., and Grütter, M.G. (2012). Human caspases in vitro: expression, purification and kinetic characterization. Protein Expr. Purif. 84, 236–246.Suche in Google Scholar

Salvesen, G.S. and Dixit, V.M. (1999). Caspase activation: the induced-proximity model. Proc. Natl. Acad. Sci. USA 96, 10964–10967.Suche in Google Scholar

Schroeder, T., Barandun, J., Flütsch, A., Briand, C., Mittl, P.R., and Grütter, M.G. (2013). Specific inhibition of caspase-3 by a competitive DARPin: molecular mimicry between native and designed inhibitors. Structure 21, 277–289.Suche in Google Scholar

Schweizer, A., Roschitzki-Voser, H., Amstutz, P., Briand, C., Gulotti-Georgieva, M., Prenosil, E., Binz, H.K., Capitani, G., Baici, A., Plückthun, A., et al. (2007). Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism. Structure 15, 625–636.Suche in Google Scholar

Seeger, M.A., Zbinden, R., Flütsch, A., Gutte, P.G., Engeler, S., Roschitzki-Voser, H., and Grütter, M.G. (2013). Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity. Protein Sci. 22, 1239–1257.Suche in Google Scholar

Sennhauser, G., Amstutz, P., Briand, C., Storchenegger, O., and Grütter, M.G. (2007). Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol. 5, e7.Suche in Google Scholar

Stefan, N., Martin-Killias, P., Wyss-Stoeckle, S., Honegger, A., Zangemeister-Wittke, U., and Plückthun, A. (2011). DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency. J. Mol. Biol. 413, 826–843.Suche in Google Scholar

Steiner, D., Forrer, P., and Plückthun, A. (2008). Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. J. Mol. Biol. 382, 1211–1227.Suche in Google Scholar

Studier, F.W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234.Suche in Google Scholar

Taylor, R.C., Cullen, S.P., and Martin, S.J. (2008). Apoptosis: controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol. 9, 231–241.Suche in Google Scholar

Watt, W., Koeplinger, K.A., Mildner, A.M., Heinrikson, R.L., Tomasselli, A.G., and Watenpaugh, K.D. (1999). The atomic-resolution structure of human caspase-8, a key activator of apoptosis. Structure 7, 1135–1143.Suche in Google Scholar

Zahnd, C., Amstutz, P., and Plückthun, A. (2007). Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Methods 4, 269–279.Suche in Google Scholar

Supplemental Material

The online version of this article (DOI: 10.1515/hsz-2014-0173) offers supplementary material, available to authorized users.

Received: 2014-3-24
Accepted: 2014-8-5
Published Online: 2014-9-5
Published in Print: 2014-10-1

©2014 by De Gruyter

Artikel in diesem Heft

  1. Frontmatter
  2. Guest Editorial
  3. Highlight: The protease web
  4. Angiotensin-I converting enzyme (ACE): structure, biological roles, and molecular basis for chloride ion dependence
  5. Non-B HIV-1 subtypes in sub-Saharan Africa: impact of subtype on protease inhibitor efficacy
  6. Inflammatory outcomes of apoptosis, necrosis and necroptosis
  7. Angiotensin-converting enzyme overexpression in myelocytes enhances the immune response
  8. The leader proteinase of foot-and-mouth disease virus: structure-function relationships in a proteolytic virulence factor
  9. Immune-modulating effects of alpha-1 antitrypsin
  10. Mammalian gamete fusion depends on the inhibition of ovastacin by fetuin-B
  11. The activity and localization patterns of cathepsins B and X in cells of the mouse gastrointestinal tract differ along its length
  12. Membrane-type I matrix metalloproteinase-dependent ectodomain shedding of mucin16/ CA-125 on ovarian cancer cells modulates adhesion and invasion of peritoneal mesothelium
  13. Homology model of human prothrombinase based on the crystal structure of Pseutarin C
  14. Specific targeting of human caspases using designed ankyrin repeat proteins
  15. Analysis of the evolution of granule associated serine proteases of immune defence (GASPIDs) suggests a revised nomenclature
https://doi.org/10.1515/hsz-2014-0173
Schlagwörter für diesen Artikel
apoptosis; cell death; DARPins; specific caspase targeting; X-ray crystallography

Artikel in diesem Heft

  1. Frontmatter
  2. Guest Editorial
  3. Highlight: The protease web
  4. Angiotensin-I converting enzyme (ACE): structure, biological roles, and molecular basis for chloride ion dependence
  5. Non-B HIV-1 subtypes in sub-Saharan Africa: impact of subtype on protease inhibitor efficacy
  6. Inflammatory outcomes of apoptosis, necrosis and necroptosis
  7. Angiotensin-converting enzyme overexpression in myelocytes enhances the immune response
  8. The leader proteinase of foot-and-mouth disease virus: structure-function relationships in a proteolytic virulence factor
  9. Immune-modulating effects of alpha-1 antitrypsin
  10. Mammalian gamete fusion depends on the inhibition of ovastacin by fetuin-B
  11. The activity and localization patterns of cathepsins B and X in cells of the mouse gastrointestinal tract differ along its length
  12. Membrane-type I matrix metalloproteinase-dependent ectodomain shedding of mucin16/ CA-125 on ovarian cancer cells modulates adhesion and invasion of peritoneal mesothelium
  13. Homology model of human prothrombinase based on the crystal structure of Pseutarin C
  14. Specific targeting of human caspases using designed ankyrin repeat proteins
  15. Analysis of the evolution of granule associated serine proteases of immune defence (GASPIDs) suggests a revised nomenclature
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