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Stabilized Human Cystatin C Variant L47C/G69C Is a Better Reporter Than the Wild-Type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases

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Abstract

Human cystatin C (HCC) binds and inhibits all types of cysteine proteases from the papain family, including cathepsins (a group of enzymes that participate in a variety of physiological processes), which are some of its natural targets. The affinities of diverse proteases for HCC, expressed as equilibrium binding constants (Kb), range from 106 to 1014 M−1. Isothermal titration calorimetry (ITC) is one of the most useful techniques to characterize the thermodynamics of molecular associations, making it possible to dissect the binding free energy into its enthalpic and entropic components. This information, together with the structural changes that occur during the different associations, could enable better understanding of the molecular basis of affinity. Notwithstanding the high sensitivity of modern calorimeters, ITC requires protein concentrations in at least the 10–100 μM range to obtain reliable data, and it is known that HCC forms oligomers in this concentration range. We present herein a comparative study of the structural, thermal stability, and oligomerization properties of HCC and its stabilized variant (sHCC) L47C/G69C (which possesses an additional disulfide bridge) as well as their binding thermodynamics to the protease chymopapain, analyzed by ITC. The results show that, because sHCC remains monomeric, it is a better reporter than wild-type HCC to characterize the thermodynamics of binding to cysteine proteases.

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  • 24 June 2019

    The original publication of this article contained a number of grammatical errors. Unfortunately, an incorrect version of the file that did not include some final language editing was inadvertently published online. The original article has been corrected.

References

  1. Turk B, Turk D, Turk V (2000) Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta 1477:98–111

    Article  CAS  PubMed  Google Scholar 

  2. Lerner UH, Grubb A (1992) Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide of malignancy. J Bone Miner Res 7:433–440

    Article  CAS  PubMed  Google Scholar 

  3. Kopitar-Jerala N (2006) The role of cystatins in cells of the immune system. FEBS Lett 580:6295–6301

    Article  CAS  PubMed  Google Scholar 

  4. Sun Q (1989) Growth stimulation of 3T3 fibroblasts by cystatin. Exp Cell Res 180:150–160

    Article  CAS  PubMed  Google Scholar 

  5. Sastre M, Calero M, Pawlik M, Mathews PM, Kumar A, Danilov V, Schmidt SD, Nixon RA, Frangione B, Levy E (2004) Binding of cystatin C to Alzheimer’s amyloid beta inhibits in vitro amyloid fibril formation. Neurobiol Aging 25:1033–1043

    Article  CAS  PubMed  Google Scholar 

  6. Palm DE, Knuckey NW, Primiano MJ, Spangenberger AG, Johanson CE (1995) Cystatin C, a protease inhibitor, in degenerating rat hippocampal neurons following transient forebrain ischemia. Brain Res 691:1–8

    Article  CAS  PubMed  Google Scholar 

  7. Janowsky R, Kozak M, Jankowska E, Grzonka Z, Grubb A, Abrahamson M, Jaskolski M (2001) Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping. Nat Struct Biol 8:316–320

    Article  CAS  Google Scholar 

  8. Abrahamson M (1996) Molecular basis for amyloidosis related to hereditary brain hemorrhage. Scand J Clin Lab Invest Suppl 226:47–56

    Article  CAS  PubMed  Google Scholar 

  9. Levy E, Sastre M, Kumar A, Gallo G, Piccardo P, Ghetti B, Tagliavini F (2001) Codeposition of cystatin C with amyloid-β protein in the brain of Alzheimer disease patients. J Neuropathol Exp Neurol 60:94–104

    Article  CAS  PubMed  Google Scholar 

  10. Nilsson M, Wang X, Rodziewicz-Motowidlo S, Janowski R, Lindström V, Önnerfjord P, Westermark G, Grzonka Z, Jaskolski M, Grubb A (2004) Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C. J Biol Chem 279:24236–24245

    Article  CAS  PubMed  Google Scholar 

  11. Kolodziejczyk R, Michalska K, Hernandez-Santoyo A, Wahlbom M, Grubb A, Jaskolski M (2010) Crystal structure of human cystatin C stabilized against amyloid formation. FEBS J 277:1726–1737

    Article  CAS  PubMed  Google Scholar 

  12. Kozak M, Jankowska E, Janowski R, Grzonka Z, Grubb A, Alvarez Fernandez M, Abrahamson M, Jaskolski M (1999) Expression of a selenomethionyl derivative and preliminary crystallographic studies of human cystatin C. Acta Crystallogr D 55:1939–1942

    Article  CAS  PubMed  Google Scholar 

  13. Wahlbom M, Wang X, Lindström V, Carlemalm E, Jaskolski M, Grubb A (2007) Fibrillogenic oligomers of human cystatin C are formed by propagated domain swapping. J Biol Chem 282:18218–18326

    Article  Google Scholar 

  14. Östner G, Lindström V, Christensen PH, Kozak M, Abrahamson M, Grubb A (2013) Stabilization, characterization, and selective removal of cystatin C amyloid oligomers. J Biol Chem 288:16438–16450

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hall A, Håkansson K, Mason RW, Grubb A, Abrahamson M (1995) Structural basis for the biological specificity of cystatin C. J Biol Chem 270:5115–5121

    Article  CAS  PubMed  Google Scholar 

  16. Björk I, Pol E, Raub-Segall E, Abrahamson M, Rowan AD, Mort JS (1994) Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes. Biochem J 299:219–225

    Article  PubMed  PubMed Central  Google Scholar 

  17. Lindahl P, Abrahamson M, Björk I (1992) Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin. Biochem J 281:49–55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Brieditis I, Raub-Segall E, Pol E, Hakansson K, Abrahamson M (1996) The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants on the inhibitor with cysteine proteinases. Biochemistry 35:10720–10726

    Article  PubMed  Google Scholar 

  19. Turk V, Stoka V, Vasiljeva O, Renko M, Sun T, Turk B, Turk D (2012) Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim Biophys Acta 1824:68–88

    Article  CAS  PubMed  Google Scholar 

  20. Wiseman T, Williston S, Brandts JF, Lin L-N (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137

    Article  CAS  PubMed  Google Scholar 

  21. Sigurskjold BW (2000) Exact analysis of competition ligand binding by displacement isothermal titration calorimetry. Anal Biochem 277:260–266

    Article  CAS  PubMed  Google Scholar 

  22. Velazquez-Campoy A, Freire E (2006) Isothermal titration calorimetry to determine association constants for high-affinity ligands. Nat Protoc 1:186–191

    Article  CAS  PubMed  Google Scholar 

  23. Buttle DJ, Abrahamson M, Barret AJ (1986) The biochemistry of the action of chymopapain in relief of sciatica. Spine 11:688–694

    Article  CAS  PubMed  Google Scholar 

  24. Björk I, Ylinenjarvi K (1990) Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin. Biochemistry 29:1770–1776

    Article  PubMed  Google Scholar 

  25. Reyes-Espinosa F, Arroyo-Reyna A, García-Gutiérrez P, Serratos IN, Zubillaga RA (2015) Effects of pH on the association between the inhibitor cystatin and the proteinase chymopapain. Protein Pept Lett 22:239–247

    Article  CAS  Google Scholar 

  26. Abrahamson M, Grubb A, Olafsson I, Lundwall Å (1987) Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C. FEBS Lett 216:229–233

    Article  CAS  PubMed  Google Scholar 

  27. Buttle DJ, Barret AJ (1984) Chymopapain. Chromatographic purification and immunological characterization. Biochem J 223:81–88

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA (2004) PDB2PQR: an automated pipeline for the setup, execution, and analysis of Poisson–Boltzmann electrostatics calculations. Nucleic Acids Res 32:W665–W667

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindhal E (2015) GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2:19–25

    Article  Google Scholar 

  30. Jorgensen WL, Maxwell DS, Tirado-Rives J (1996) Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J Am Chem Soc 118:11225–11236

    Article  CAS  Google Scholar 

  31. Hess B, Bekker H, Berendsen HJC, Fraaije JGEM (1997) LINCS: a linear constraint solver for molecular simulations. J Comput Chem 18:1463–1472

    Article  CAS  Google Scholar 

  32. Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. J Chem Phys 98:10089–10092

    Article  CAS  Google Scholar 

  33. Carrette O, Burkhard PR, Hughes S, Hochstrasser DF, Sanchez JC (2005) Truncated cystatin C in cerebrospinal fluid: technical artefact or biological process? Proteomics 5:3060–3065

    Article  PubMed  Google Scholar 

  34. Del Boccio P, Pieragostino D, Lugaresi A, Di Ioia M, Pavone B, Travaglini D, D’Aguanno S, Bernardini S, Sacchetta P, Federici G, Di Ilio C, Gambi D, Urbani A (2007) Cleavage of cystatin C is not associated with multiple sclerosis. Ann Neurol 62:201–204

    Article  CAS  PubMed  Google Scholar 

  35. Perez-Iratxeta C, Andrade-Navarro MA (2008) K2D2: estimation of protein secondary structure from circular dichroism spectra. BMC Struct Biol 8:25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Calvete JJ (2007) Determinación del número de grupos sulfidrilo y de enlaces disulfuro mediante espectrometría de masas. Proteómica 0:21–28. https://helvia.uco.es/bitstream/handle/10396/8926/pro4.pdf?sequence=1&isAllowed=y

  37. Marky LA, Breslauer KJ (1987) Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26:1601–1620

    Article  CAS  PubMed  Google Scholar 

  38. Kuzmanic A, Zagrovic B (2010) Determination of ensemble-average pairwise root mean-square deviation from experimental B-factors. Biophys J 98:861–871

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Perlenfein TJ, Murphy RM (2016) Expression, purification, and characterization of human cystatin C monomers and oligomers. Protein Expr Purif 117:35–43

    Article  CAS  PubMed  Google Scholar 

  40. Žerovnik E, Cimerman N, Kos J, Turk V, Lohner K (1997) Thermal denaturation of human cystatin C and two of its variants; comparison to chicken cystatin. Biol Chem 378:1199–1203

    PubMed  Google Scholar 

  41. Pace CN, Grimsley GR, Thomson JA, Barnet BJ (1988) Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds. J Biol Chem 263:11820–11825

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Council of Science and Technology (CONACyT, México) by grants no. 181049 and 237256, by scholarship no. 283296 to D.O. Tovar-Anaya, and by a postdoctoral scholarship to M.T. Vieyra-Eusebio. The authors thank the Laboratorio de Supercómputo y Visualización en Paralelo and the Laboratorio Divisional de Espectrometría de Masas at the Universidad Autónoma Metropolitana-Iztapalapa for the use of their facilities. We are grateful to Miguel Costas from the Facultad de Química, UNAM for providing the VP‐DSC capillary facility to perform the differential scanning calorimetry experiments. We also thank Georgina Garza-Ramos from the Facultad de Medicina, UNAM for her expert help and facilitating our use of the FPLC for SEC experiments.

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  1. Departamento de Química, Universidad Autónoma Metropolitana- Iztapalapa, Ciudad De México, 09340, Mexico

    David O. Tovar-Anaya, L. Irais Vera-Robles, M. Teresa Vieyra-Eusebio, Ponciano García-Gutiérrez, Francisco Reyes-Espinosa, Andrés Hernández-Arana, J. Alfonso Arroyo-Reyna & Rafael A. Zubillaga

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  1. David O. Tovar-Anaya
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Correspondence to Rafael A. Zubillaga.

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The original version of this article was revised: The grammatical errors in the article have been corrected.

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Tovar-Anaya, D.O., Vera-Robles, L.I., Vieyra-Eusebio, M.T. et al. Stabilized Human Cystatin C Variant L47C/G69C Is a Better Reporter Than the Wild-Type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases. Protein J 38, 598–607 (2019). https://doi.org/10.1007/s10930-019-09839-2

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