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. 2000 Sep;9(9):1700–1708. doi: 10.1110/ps.9.9.1700

Protein engineering as a strategy to avoid formation of amyloid fibrils.

V Villegas 1, J Zurdo 1, V V Filimonov 1, F X Avilés 1, C M Dobson 1, L Serrano 1
PMCID: PMC2144697  PMID: 11045616

Abstract

The activation domain of human procarboxypeptidase A2 (ADA2h) aggregates following thermal or chemical denaturation at acidic pH. The aggregated material contains well-defined ordered structures with all the characteristics of the fibrils associated with amyloidotic diseases. Variants of ADA2h containing a series of mutations designed to increase the local stability of each of the two helical regions of the protein have been found to have a substantially reduced propensity to form fibrils. This arises from a reduced tendency of the denatured species to aggregate rather than from a change in the overall stability of the native state. The reduction in aggregation propensity may result from an increase in the stability of local relative to longer range interactions within the polypeptide chain. These findings show that the intrinsic ability of a protein to form amyloid can be altered substantially by protein engineering methods without perturbing significantly its overall stability or activity. This suggests new strategies for combating diseases associated with the formation of aggregated proteins and for the design of novel protein or peptide therapeutics.

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Selected References

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  1. Aloy P., Catasús L., Villegas V., Reverter D., Vendrell J., Avilés F. X. Comparative analysis of the sequences and three-dimensional models of human procarboxypeptidases A1, A2 and B. Biol Chem. 1998 Feb;379(2):149–155. doi: 10.1515/bchm.1998.379.2.149. [DOI] [PubMed] [Google Scholar]
  2. Arvinte T., Cudd A., Drake A. F. The structure and mechanism of formation of human calcitonin fibrils. J Biol Chem. 1993 Mar 25;268(9):6415–6422. [PubMed] [Google Scholar]
  3. Bauer H. H., Aebi U., Häner M., Hermann R., Müller M., Merkle H. P. Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin. J Struct Biol. 1995 Jul-Aug;115(1):1–15. doi: 10.1006/jsbi.1995.1024. [DOI] [PubMed] [Google Scholar]
  4. Blake C., Serpell L. Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous beta-sheet helix. Structure. 1996 Aug 15;4(8):989–998. doi: 10.1016/s0969-2126(96)00104-9. [DOI] [PubMed] [Google Scholar]
  5. Booth D. R., Sunde M., Bellotti V., Robinson C. V., Hutchinson W. L., Fraser P. E., Hawkins P. N., Dobson C. M., Radford S. E., Blake C. C. Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature. 1997 Feb 27;385(6619):787–793. doi: 10.1038/385787a0. [DOI] [PubMed] [Google Scholar]
  6. Canet D., Sunde M., Last A. M., Miranker A., Spencer A., Robinson C. V., Dobson C. M. Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants. Biochemistry. 1999 May 18;38(20):6419–6427. doi: 10.1021/bi983037t. [DOI] [PubMed] [Google Scholar]
  7. Catasús L., Vendrell J., Avilés F. X., Carreira S., Puigserver A., Billeter M. The sequence and conformation of human pancreatic procarboxypeptidase A2. cDNA cloning, sequence analysis, and three-dimensional model. J Biol Chem. 1995 Mar 24;270(12):6651–6657. doi: 10.1074/jbc.270.12.6651. [DOI] [PubMed] [Google Scholar]
  8. Chen Y. H., Yang J. T., Chau K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. doi: 10.1021/bi00713a027. [DOI] [PubMed] [Google Scholar]
  9. Chiti F., Webster P., Taddei N., Clark A., Stefani M., Ramponi G., Dobson C. M. Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3590–3594. doi: 10.1073/pnas.96.7.3590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen F. E., Prusiner S. B. Pathologic conformations of prion proteins. Annu Rev Biochem. 1998;67:793–819. doi: 10.1146/annurev.biochem.67.1.793. [DOI] [PubMed] [Google Scholar]
  11. Dobson C. M. Protein misfolding, evolution and disease. Trends Biochem Sci. 1999 Sep;24(9):329–332. doi: 10.1016/s0968-0004(99)01445-0. [DOI] [PubMed] [Google Scholar]
  12. Fabian H., Szendrei G. I., Mantsch H. H., Otvos L., Jr Comparative analysis of human and Dutch-type Alzheimer beta-amyloid peptides by infrared spectroscopy and circular dichroism. Biochem Biophys Res Commun. 1993 Feb 26;191(1):232–239. doi: 10.1006/bbrc.1993.1207. [DOI] [PubMed] [Google Scholar]
  13. Fraser P. E., Nguyen J. T., Inouye H., Surewicz W. K., Selkoe D. J., Podlisny M. B., Kirschner D. A. Fibril formation by primate, rodent, and Dutch-hemorrhagic analogues of Alzheimer amyloid beta-protein. Biochemistry. 1992 Nov 10;31(44):10716–10723. doi: 10.1021/bi00159a011. [DOI] [PubMed] [Google Scholar]
  14. García-Sáez I., Reverter D., Vendrell J., Avilés F. X., Coll M. The three-dimensional structure of human procarboxypeptidase A2. Deciphering the basis of the inhibition, activation and intrinsic activity of the zymogen. EMBO J. 1997 Dec 1;16(23):6906–6913. doi: 10.1093/emboj/16.23.6906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gasset M., Baldwin M. A., Lloyd D. H., Gabriel J. M., Holtzman D. M., Cohen F., Fletterick R., Prusiner S. B. Predicted alpha-helical regions of the prion protein when synthesized as peptides form amyloid. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10940–10944. doi: 10.1073/pnas.89.22.10940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ghetti B., Piccardo P., Frangione B., Bugiani O., Giaccone G., Young K., Prelli F., Farlow M. R., Dlouhy S. R., Tagliavini F. Prion protein amyloidosis. Brain Pathol. 1996 Apr;6(2):127–145. doi: 10.1111/j.1750-3639.1996.tb00796.x. [DOI] [PubMed] [Google Scholar]
  17. Guijarro J. I., Sunde M., Jones J. A., Campbell I. D., Dobson C. M. Amyloid fibril formation by an SH3 domain. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4224–4228. doi: 10.1073/pnas.95.8.4224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Harper J. D., Lansbury P. T., Jr Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu Rev Biochem. 1997;66:385–407. doi: 10.1146/annurev.biochem.66.1.385. [DOI] [PubMed] [Google Scholar]
  19. Hilbich C., Kisters-Woike B., Reed J., Masters C. L., Beyreuther K. Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer's disease beta A4 peptides. J Mol Biol. 1992 Nov 20;228(2):460–473. doi: 10.1016/0022-2836(92)90835-8. [DOI] [PubMed] [Google Scholar]
  20. Hurle M. R., Helms L. R., Li L., Chan W., Wetzel R. A role for destabilizing amino acid replacements in light-chain amyloidosis. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5446–5450. doi: 10.1073/pnas.91.12.5446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jarrett J. T., Lansbury P. T., Jr Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB. Biochemistry. 1992 Dec 15;31(49):12345–12352. doi: 10.1021/bi00164a008. [DOI] [PubMed] [Google Scholar]
  22. Jarrett J. T., Lansbury P. T., Jr Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell. 1993 Jun 18;73(6):1055–1058. doi: 10.1016/0092-8674(93)90635-4. [DOI] [PubMed] [Google Scholar]
  23. Jiménez J. L., Guijarro J. I., Orlova E., Zurdo J., Dobson C. M., Sunde M., Saibil H. R. Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing. EMBO J. 1999 Feb 15;18(4):815–821. doi: 10.1093/emboj/18.4.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  25. Kelly J. W. Amyloid fibril formation and protein misassembly: a structural quest for insights into amyloid and prion diseases. Structure. 1997 May 15;5(5):595–600. doi: 10.1016/s0969-2126(97)00215-3. [DOI] [PubMed] [Google Scholar]
  26. Kelly J. W. The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr Opin Struct Biol. 1998 Feb;8(1):101–106. doi: 10.1016/s0959-440x(98)80016-x. [DOI] [PubMed] [Google Scholar]
  27. Klunk W. E., Pettegrew J. W., Abraham D. J. Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J Histochem Cytochem. 1989 Aug;37(8):1273–1281. doi: 10.1177/37.8.2666510. [DOI] [PubMed] [Google Scholar]
  28. Klunk W. E., Pettegrew J. W., Abraham D. J. Two simple methods for quantifying low-affinity dye-substrate binding. J Histochem Cytochem. 1989 Aug;37(8):1293–1297. doi: 10.1177/37.8.2666512. [DOI] [PubMed] [Google Scholar]
  29. Krimm S., Bandekar J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv Protein Chem. 1986;38:181–364. doi: 10.1016/s0065-3233(08)60528-8. [DOI] [PubMed] [Google Scholar]
  30. Lai Z., Colón W., Kelly J. W. The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid. Biochemistry. 1996 May 21;35(20):6470–6482. doi: 10.1021/bi952501g. [DOI] [PubMed] [Google Scholar]
  31. LeVine H., 3rd Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 1993 Mar;2(3):404–410. doi: 10.1002/pro.5560020312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Liemann S., Glockshuber R. Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein. Biochemistry. 1999 Mar 16;38(11):3258–3267. doi: 10.1021/bi982714g. [DOI] [PubMed] [Google Scholar]
  33. Litvinovich S. V., Brew S. A., Aota S., Akiyama S. K., Haudenschild C., Ingham K. C. Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module. J Mol Biol. 1998 Jul 10;280(2):245–258. doi: 10.1006/jmbi.1998.1863. [DOI] [PubMed] [Google Scholar]
  34. McCutchen S. L., Lai Z., Miroy G. J., Kelly J. W., Colón W. Comparison of lethal and nonlethal transthyretin variants and their relationship to amyloid disease. Biochemistry. 1995 Oct 17;34(41):13527–13536. doi: 10.1021/bi00041a032. [DOI] [PubMed] [Google Scholar]
  35. Merlini G., Ascari E., Amboldi N., Bellotti V., Arbustini E., Perfetti V., Ferrari M., Zorzoli I., Marinone M. G., Garini P. Interaction of the anthracycline 4'-iodo-4'-deoxydoxorubicin with amyloid fibrils: inhibition of amyloidogenesis. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2959–2963. doi: 10.1073/pnas.92.7.2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Minor D. L., Jr, Kim P. S. Context is a major determinant of beta-sheet propensity. Nature. 1994 Sep 15;371(6494):264–267. doi: 10.1038/371264a0. [DOI] [PubMed] [Google Scholar]
  37. Minor D. L., Jr, Kim P. S. Measurement of the beta-sheet-forming propensities of amino acids. Nature. 1994 Feb 17;367(6464):660–663. doi: 10.1038/367660a0. [DOI] [PubMed] [Google Scholar]
  38. Moriarty D. F., Raleigh D. P. Effects of sequential proline substitutions on amyloid formation by human amylin20-29. Biochemistry. 1999 Feb 9;38(6):1811–1818. doi: 10.1021/bi981658g. [DOI] [PubMed] [Google Scholar]
  39. Moriarty D. F., Vagts S., Raleigh D. P. A role for the C-terminus of calcitonin in aggregation and gel formation: a comparative study of C-terminal fragments of human and salmon calcitonin. Biochem Biophys Res Commun. 1998 Apr 17;245(2):344–348. doi: 10.1006/bbrc.1998.8425. [DOI] [PubMed] [Google Scholar]
  40. Riek R., Wider G., Billeter M., Hornemann S., Glockshuber R., Wüthrich K. Prion protein NMR structure and familial human spongiform encephalopathies. Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11667–11672. doi: 10.1073/pnas.95.20.11667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Salomon A. R., Marcinowski K. J., Friedland R. P., Zagorski M. G. Nicotine inhibits amyloid formation by the beta-peptide. Biochemistry. 1996 Oct 22;35(42):13568–13578. doi: 10.1021/bi9617264. [DOI] [PubMed] [Google Scholar]
  42. Seilheimer B., Bohrmann B., Bondolfi L., Müller F., Stüber D., Döbeli H. The toxicity of the Alzheimer's beta-amyloid peptide correlates with a distinct fiber morphology. J Struct Biol. 1997 Jun;119(1):59–71. doi: 10.1006/jsbi.1997.3859. [DOI] [PubMed] [Google Scholar]
  43. Soto C., Castaño E. M., Frangione B., Inestrosa N. C. The alpha-helical to beta-strand transition in the amino-terminal fragment of the amyloid beta-peptide modulates amyloid formation. J Biol Chem. 1995 Feb 17;270(7):3063–3067. doi: 10.1074/jbc.270.7.3063. [DOI] [PubMed] [Google Scholar]
  44. Soto C., Castaño E. M. The conformation of Alzheimer's beta peptide determines the rate of amyloid formation and its resistance to proteolysis. Biochem J. 1996 Mar 1;314(Pt 2):701–707. doi: 10.1042/bj3140701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Soto C., Kindy M. S., Baumann M., Frangione B. Inhibition of Alzheimer's amyloidosis by peptides that prevent beta-sheet conformation. Biochem Biophys Res Commun. 1996 Sep 24;226(3):672–680. doi: 10.1006/bbrc.1996.1413. [DOI] [PubMed] [Google Scholar]
  46. Sunde M., Serpell L. C., Bartlam M., Fraser P. E., Pepys M. B., Blake C. C. Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J Mol Biol. 1997 Oct 31;273(3):729–739. doi: 10.1006/jmbi.1997.1348. [DOI] [PubMed] [Google Scholar]
  47. Surewicz W. K., Mantsch H. H., Chapman D. Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment. Biochemistry. 1993 Jan 19;32(2):389–394. doi: 10.1021/bi00053a001. [DOI] [PubMed] [Google Scholar]
  48. Swietnicki W., Petersen R. B., Gambetti P., Surewicz W. K. Familial mutations and the thermodynamic stability of the recombinant human prion protein. J Biol Chem. 1998 Nov 20;273(47):31048–31052. doi: 10.1074/jbc.273.47.31048. [DOI] [PubMed] [Google Scholar]
  49. Tagliavini F., McArthur R. A., Canciani B., Giaccone G., Porro M., Bugiani M., Lievens P. M., Bugiani O., Peri E., Dall'Ara P. Effectiveness of anthracycline against experimental prion disease in Syrian hamsters. Science. 1997 May 16;276(5315):1119–1122. doi: 10.1126/science.276.5315.1119. [DOI] [PubMed] [Google Scholar]
  50. Tan S. Y., Pepys M. B. Amyloidosis. Histopathology. 1994 Nov;25(5):403–414. doi: 10.1111/j.1365-2559.1994.tb00001.x. [DOI] [PubMed] [Google Scholar]
  51. Turnell W. G., Finch J. T. Binding of the dye congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences. J Mol Biol. 1992 Oct 20;227(4):1205–1223. doi: 10.1016/0022-2836(92)90532-o. [DOI] [PubMed] [Google Scholar]
  52. Viguera A. R., Villegas V., Avilés F. X., Serrano L. Favourable native-like helical local interactions can accelerate protein folding. Fold Des. 1997;2(1):23–33. doi: 10.1016/S1359-0278(97)00003-5. [DOI] [PubMed] [Google Scholar]
  53. Villegas V., Azuaga A., Catasús L., Reverter D., Mateo P. L., Avilés F. X., Serrano L. Evidence for a two-state transition in the folding process of the activation domain of human procarboxypeptidase A2. Biochemistry. 1995 Nov 21;34(46):15105–15110. doi: 10.1021/bi00046a017. [DOI] [PubMed] [Google Scholar]
  54. Villegas V., Martínez J. C., Avilés F. X., Serrano L. Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain. J Mol Biol. 1998 Nov 13;283(5):1027–1036. doi: 10.1006/jmbi.1998.2158. [DOI] [PubMed] [Google Scholar]
  55. Villegas V., Viguera A. R., Avilés F. X., Serrano L. Stabilization of proteins by rational design of alpha-helix stability using helix/coil transition theory. Fold Des. 1996;1(1):29–34. [PubMed] [Google Scholar]
  56. Westermark P., Engström U., Johnson K. H., Westermark G. T., Betsholtz C. Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5036–5040. doi: 10.1073/pnas.87.13.5036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wood S. J., MacKenzie L., Maleeff B., Hurle M. R., Wetzel R. Selective inhibition of Abeta fibril formation. J Biol Chem. 1996 Feb 23;271(8):4086–4092. doi: 10.1074/jbc.271.8.4086. [DOI] [PubMed] [Google Scholar]
  58. Wood S. J., Wetzel R., Martin J. D., Hurle M. R. Prolines and amyloidogenicity in fragments of the Alzheimer's peptide beta/A4. Biochemistry. 1995 Jan 24;34(3):724–730. doi: 10.1021/bi00003a003. [DOI] [PubMed] [Google Scholar]

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