Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Jan 1;116(1):157–166. doi: 10.1083/jcb.116.1.157

A synthetic peptide representing the consensus sequence motif at the carboxy-terminal end of the rod domain inhibits intermediate filament assembly and disassembles preformed filaments

PMCID: PMC2289271  PMID: 1370491

Abstract

All intermediate filament (IF) proteins share a highly conserved sequence motif at the COOH-terminal end of their rod domains. We have studied the influence of a 20-residue peptide, representing the consensus motif on filament formation and stability. Addition of the peptide at a 10-20-fold molar excess over keratins K8 plus K18 had a severe effect on subsequent IF assembly. Filaments displayed a rough surface and variable diameters with a substantial amount present in unravelled form. At higher peptide concentration (50-100-fold molar excess), IF formation was completely inhibited and instead only loose aggregates of "globular" particles were formed. The peptide also influenced performed keratin IF in a dose-dependent manner. While a three-fold molar excess was sufficient to cause partial fragmentation of IF, a 50-fold molar excess caused complete disassembly within 5 min. Loosely associated protofibrils, short needlelike IF fragments, and aggregates of globular particles were detected. The motif peptide also caused the disassembly of filaments formed by desmin, a type III IF protein. Peptide concentrations and incubation times required for complete disassembly were somewhat higher than for the filaments containing K8 plus K18. A 50-fold molar excess was sufficient to cause complete disassembly within 1 h. Peptides unrelated in sequence to the motif did not interfere with filament formation or stability even when present for more than 12 h at a 100-fold molar excess. The results suggest that the motif sequence normally binds to a specific acceptor site for which the motif peptide can successfully compete. Taken together with current models of IF structure the results indicate that normal binding of the motif sequence to its acceptor must play an essential role in IF formation, possibly by directing the proper alignment of neighboring tetramers or protofilaments. Finally we show that in vitro formed IF are much more sensitive and dynamic strutures than previously thought.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aebi U., Fowler W. E., Rew P., Sun T. T. The fibrillar substructure of keratin filaments unraveled. J Cell Biol. 1983 Oct;97(4):1131–1143. doi: 10.1083/jcb.97.4.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albers K., Fuchs E. Expression of mutant keratin cDNAs in epithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments. J Cell Biol. 1989 Apr;108(4):1477–1493. doi: 10.1083/jcb.108.4.1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Albers K., Fuchs E. The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines. J Cell Biol. 1987 Aug;105(2):791–806. doi: 10.1083/jcb.105.2.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Becker T., Weber K., Johnsson N. Protein-protein recognition via short amphiphilic helices; a mutational analysis of the binding site of annexin II for p11. EMBO J. 1990 Dec;9(13):4207–4213. doi: 10.1002/j.1460-2075.1990.tb07868.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birkenberger L., Ip W. Properties of the desmin tail domain: studies using synthetic peptides and antipeptide antibodies. J Cell Biol. 1990 Nov;111(5 Pt 1):2063–2075. doi: 10.1083/jcb.111.5.2063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Coulombe P. A., Chan Y. M., Albers K., Fuchs E. Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro. J Cell Biol. 1990 Dec;111(6 Pt 2):3049–3064. doi: 10.1083/jcb.111.6.3049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coulombe P. A., Fuchs E. Elucidating the early stages of keratin filament assembly. J Cell Biol. 1990 Jul;111(1):153–169. doi: 10.1083/jcb.111.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eichner R., Rew P., Engel A., Aebi U. Human epidermal keratin filaments: studies on their structure and assembly. Ann N Y Acad Sci. 1985;455:381–402. doi: 10.1111/j.1749-6632.1985.tb50424.x. [DOI] [PubMed] [Google Scholar]
  10. Engel A., Eichner R., Aebi U. Polymorphism of reconstituted human epidermal keratin filaments: determination of their mass-per-length and width by scanning transmission electron microscopy (STEM). J Ultrastruct Res. 1985 Mar;90(3):323–335. doi: 10.1016/s0022-5320(85)80010-1. [DOI] [PubMed] [Google Scholar]
  11. Fisher D. Z., Chaudhary N., Blobel G. cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6450–6454. doi: 10.1073/pnas.83.17.6450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geisler N., Kaufmann E., Weber K. Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments. J Mol Biol. 1985 Mar 5;182(1):173–177. doi: 10.1016/0022-2836(85)90035-x. [DOI] [PubMed] [Google Scholar]
  13. Geisler N., Kaufmann E., Weber K. Proteinchemical characterization of three structurally distinct domains along the protofilament unit of desmin 10 nm filaments. Cell. 1982 Aug;30(1):277–286. doi: 10.1016/0092-8674(82)90033-2. [DOI] [PubMed] [Google Scholar]
  14. Geisler N., Weber K. Purification of smooth-muscle desmin and a protein-chemical comparison of desmins from chicken gizzard and hog stomach. Eur J Biochem. 1980 Oct;111(2):425–433. doi: 10.1111/j.1432-1033.1980.tb04957.x. [DOI] [PubMed] [Google Scholar]
  15. Geisler N., Weber K. The amino acid sequence of chicken muscle desmin provides a common structural model for intermediate filament proteins. EMBO J. 1982;1(12):1649–1656. doi: 10.1002/j.1460-2075.1982.tb01368.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gerke V. Consensus peptide antibodies reveal a widespread occurrence of Ca2+/lipid-binding proteins of the annexin family. FEBS Lett. 1989 Dec 4;258(2):259–262. doi: 10.1016/0014-5793(89)81668-0. [DOI] [PubMed] [Google Scholar]
  17. Gill S. R., Wong P. C., Monteiro M. J., Cleveland D. W. Assembly properties of dominant and recessive mutations in the small mouse neurofilament (NF-L) subunit. J Cell Biol. 1990 Nov;111(5 Pt 1):2005–2019. doi: 10.1083/jcb.111.5.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hanukoglu I., Fuchs E. The cDNA sequence of a Type II cytoskeletal keratin reveals constant and variable structural domains among keratins. Cell. 1983 Jul;33(3):915–924. doi: 10.1016/0092-8674(83)90034-x. [DOI] [PubMed] [Google Scholar]
  19. Hatzfeld M., Franke W. W. Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol. 1985 Nov;101(5 Pt 1):1826–1841. doi: 10.1083/jcb.101.5.1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hatzfeld M., Weber K. Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain. J Cell Sci. 1991 Jun;99(Pt 2):351–362. doi: 10.1242/jcs.99.2.351. [DOI] [PubMed] [Google Scholar]
  21. Hatzfeld M., Weber K. Tailless keratins assemble into regular intermediate filaments in vitro. J Cell Sci. 1990 Oct;97(Pt 2):317–324. doi: 10.1242/jcs.97.2.317. [DOI] [PubMed] [Google Scholar]
  22. Henderson D., Geisler N., Weber K. A periodic ultrastructure in intermediate filaments. J Mol Biol. 1982 Feb 25;155(2):173–176. doi: 10.1016/0022-2836(82)90444-2. [DOI] [PubMed] [Google Scholar]
  23. Huber R., Römisch J., Paques E. P. The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes. EMBO J. 1990 Dec;9(12):3867–3874. doi: 10.1002/j.1460-2075.1990.tb07605.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ip W., Hartzer M. K., Pang Y. Y., Robson R. M. Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments. J Mol Biol. 1985 Jun 5;183(3):365–375. doi: 10.1016/0022-2836(85)90007-5. [DOI] [PubMed] [Google Scholar]
  25. Kaufmann E., Weber K., Geisler N. Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues. J Mol Biol. 1985 Oct 20;185(4):733–742. doi: 10.1016/0022-2836(85)90058-0. [DOI] [PubMed] [Google Scholar]
  26. Lu X., Lane E. B. Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation. Cell. 1990 Aug 24;62(4):681–696. doi: 10.1016/0092-8674(90)90114-t. [DOI] [PubMed] [Google Scholar]
  27. Magin T. M., Hatzfeld M., Franke W. W. Analysis of cytokeratin domains by cloning and expression of intact and deleted polypeptides in Escherichia coli. EMBO J. 1987 Sep;6(9):2607–2615. doi: 10.1002/j.1460-2075.1987.tb02551.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McKeon F. D., Kirschner M. W., Caput D. Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature. 1986 Feb 6;319(6053):463–468. doi: 10.1038/319463a0. [DOI] [PubMed] [Google Scholar]
  29. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  30. Ngai J., Coleman T. R., Lazarides E. Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly. Cell. 1990 Feb 9;60(3):415–427. doi: 10.1016/0092-8674(90)90593-4. [DOI] [PubMed] [Google Scholar]
  31. Osborn M., Weber K. Tumor diagnosis by intermediate filament typing: a novel tool for surgical pathology. Lab Invest. 1983 Apr;48(4):372–394. [PubMed] [Google Scholar]
  32. Parry D. A., Steven A. C., Steinert P. M. The coiled-coil molecules of intermediate filaments consist of two parallel chains in exact axial register. Biochem Biophys Res Commun. 1985 Mar 29;127(3):1012–1018. doi: 10.1016/s0006-291x(85)80045-0. [DOI] [PubMed] [Google Scholar]
  33. Potschka M., Nave R., Weber K., Geisler N. The two coiled coils in the isolated rod domain of the intermediate filament protein desmin are staggered. A hydrodynamic analysis of tetramers and dimers. Eur J Biochem. 1990 Jul 5;190(3):503–508. doi: 10.1111/j.1432-1033.1990.tb15602.x. [DOI] [PubMed] [Google Scholar]
  34. Pruss R. M., Mirsky R., Raff M. C., Thorpe R., Dowding A. J., Anderton B. H. All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell. 1981 Dec;27(3 Pt 2):419–428. doi: 10.1016/0092-8674(81)90383-4. [DOI] [PubMed] [Google Scholar]
  35. Quax W., Egberts W. V., Hendriks W., Quax-Jeuken Y., Bloemendal H. The structure of the vimentin gene. Cell. 1983 Nov;35(1):215–223. doi: 10.1016/0092-8674(83)90224-6. [DOI] [PubMed] [Google Scholar]
  36. Quinlan R. A., Cohlberg J. A., Schiller D. L., Hatzfeld M., Franke W. W. Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells. J Mol Biol. 1984 Sep 15;178(2):365–388. doi: 10.1016/0022-2836(84)90149-9. [DOI] [PubMed] [Google Scholar]
  37. Quinlan R. A., Hatzfeld M., Franke W. W., Lustig A., Schulthess T., Engel J. Characterization of dimer subunits of intermediate filament proteins. J Mol Biol. 1986 Nov 20;192(2):337–349. doi: 10.1016/0022-2836(86)90369-4. [DOI] [PubMed] [Google Scholar]
  38. Quinlan R. A., Schiller D. L., Hatzfeld M., Achtstätter T., Moll R., Jorcano J. L., Magin T. M., Franke W. W. Patterns of expression and organization of cytokeratin intermediate filaments. Ann N Y Acad Sci. 1985;455:282–306. doi: 10.1111/j.1749-6632.1985.tb50418.x. [DOI] [PubMed] [Google Scholar]
  39. Raats J. M., Pieper F. R., Vree Egberts W. T., Verrijp K. N., Ramaekers F. C., Bloemendal H. Assembly of amino-terminally deleted desmin in vimentin-free cells. J Cell Biol. 1990 Nov;111(5 Pt 1):1971–1985. doi: 10.1083/jcb.111.5.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Steinert P. M., Rice R. H., Roop D. R., Trus B. L., Steven A. C. Complete amino acid sequence of a mouse epidermal keratin subunit and implications for the structure of intermediate filaments. Nature. 1983 Apr 28;302(5911):794–800. doi: 10.1038/302794a0. [DOI] [PubMed] [Google Scholar]
  41. Steinert P. M., Roop D. R. Molecular and cellular biology of intermediate filaments. Annu Rev Biochem. 1988;57:593–625. doi: 10.1146/annurev.bi.57.070188.003113. [DOI] [PubMed] [Google Scholar]
  42. Steinert P. M., Steven A. C., Roop D. R. The molecular biology of intermediate filaments. Cell. 1985 Sep;42(2):411–420. doi: 10.1016/0092-8674(85)90098-4. [DOI] [PubMed] [Google Scholar]
  43. Steinert P. M. The two-chain coiled-coil molecule of native epidermal keratin intermediate filaments is a type I-type II heterodimer. J Biol Chem. 1990 May 25;265(15):8766–8774. [PubMed] [Google Scholar]
  44. Steven A. C., Hainfeld J. F., Trus B. L., Wall J. S., Steinert P. M. Epidermal keratin filaments assembled in vitro have masses-per-unit-length that scale according to average subunit mass: structural basis for homologous packing of subunits in intermediate filaments. J Cell Biol. 1983 Dec;97(6):1939–1944. doi: 10.1083/jcb.97.6.1939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stewart M., Quinlan R. A., Moir R. D. Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure. J Cell Biol. 1989 Jul;109(1):225–234. doi: 10.1083/jcb.109.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vikstrom K. L., Borisy G. G., Goldman R. D. Dynamic aspects of intermediate filament networks in BHK-21 cells. Proc Natl Acad Sci U S A. 1989 Jan;86(2):549–553. doi: 10.1073/pnas.86.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weber K., Plessmann U., Dodemont H., Kossmagk-Stephan K. Amino acid sequences and homopolymer-forming ability of the intermediate filament proteins from an invertebrate epithelium. EMBO J. 1988 Oct;7(10):2995–3001. doi: 10.1002/j.1460-2075.1988.tb03162.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wong P. C., Cleveland D. W. Characterization of dominant and recessive assembly-defective mutations in mouse neurofilament NF-M. J Cell Biol. 1990 Nov;111(5 Pt 1):1987–2003. doi: 10.1083/jcb.111.5.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES