Molecular mechanisms of amyloid self-regulation

Amyloid is associated with both pathological protein deposits and the formation of functional protein structures. Therefore, several strategies have evolved to control the formation or inhibition of amyloid in vivo. In this thesis, three separate systems were investigated in which amyloidogenic protein segments are coupled to regulatory elements that prevent or promote fibrillation. We describe the molecular mechanism for how (a) a propeptide segment prevents the uncontrolled aggregation of the mature peptide, (b) a chaperone domain inhibits amyloid formation, and (c) a pH-dependent relay controls protein assembly. For this purpose, mass spectrometry (MS)-based approaches to structural biology were applied and extended, involving gas phase interaction studies and hydrogen/deuterium exchange MS.

(a) Proinsulin C-peptide is beneficial for the preservation of insulin activity. We show that C-peptide interferes with insulin amyloid fibril formation at low pH and how conserved glutamate residues in C-peptide mediate reversible co-precipitation with insulin. A mechanism is proposed for how the balance between zinc and C-peptide mediates sorting of insulin into slow acting and rapid acting forms inside the secretory granules of the pancreatic -cells, which potentially links C-peptide to diabetes type 1 and 2.

(b) Lung surfactant protein C (SP-C) is a highly amyloidogenic transmembrane polypeptide that controls surface tension in the alveolar phospholipid bilayer. Its proprotein includes a conserved chaperone domain termed BRICHOS, which is also associated with neurodegenerative disorders. It is shown here how BRICHOS and its N-terminal linker recognize hydrophobic residues and trap the SP-C segment in a - hairpin conformation to prevent amyloid formation.

(c) Spider silk is synthesized as a highly soluble protein that assembles into silk in a pH-dependent fashion. It is shown that the spider silk protein N-terminal (NT) domain dimerizes at the same pH interval that triggers silk assembly, and we define the associated structural changes. Furthermore, the use of the NT domain as a solubility tag for the expression of aggregation-prone proteins is demonstrated.

In summary, we have determined the molecular basis for three distinct mechanisms by which fibril formation is controlled through autoregulatory elements and provide insights into nature’s strategies to control amyloid formation and prevention. Based on these findings, we can now make conclusions about nature’s handling of amyloidogenic proteins and their function in general.

List of scientific papers

I. Nerelius, C., Fitzen, M., Johansson, J. (2010) Amino acid sequence determinants and molecular chaperones in amyloid fibril formation. Biochem Biophys Res Commun. 396: 2-6.
https://doi.org/10.1016/j.bbrc.2010.02.105

II. Landreh, M., Astorga-Wells, J., Johansson, J., Bergman, T., Jörnvall, H. (2011) New developments in protein structure and function analysis by mass spectrometry and use of H/D exchange microfluidics. FEBS J. 278: 3815–21.
https://doi.org/10.1111/j.1742-4658.2011.08215.x

III. Astorga-Wells, J., Landreh, M., Bergman, T., Jörnvall, H. (2011). A membrane cell for on-line Hydrogen/Deuterium exchange to study protein folding and protein-protein interaction by mass spectrometry. Mol Cell Proteomics. 10: M110.006510.
https://doi.org/10.1074/mcp.M110.006510

IV. Landreh, M., Stukenborg, J.-B., Willander, H., Söder, O., Johansson, J., Jörnvall, H. (2012) Proinsulin C-peptide interferes with insulin fibril formation. Biochem Biophys Res Commun. 418: 489-93.
https://doi.org/10.1016/j.bbrc.2012.01.051

V. Landreh, M., Alvelius, G., Willander, H., Stukenborg, J.-B., Söder, O., Johansson, J., Jörnvall, H. (2012) Insulin solubility transitions by pHdependent interactions with proinsulin C-peptide. FEBS J. [Accepted]
https://doi.org/10.1111/febs.12045

VI. Landreh, M., Johansson, J., Jörnvall, H. Interactions of proinsulin Cpeptide in the beta cell secretory granule: A molecular balance with implications for diabetes-associated conditions. [Submitted]

VII. Fitzen, M., Alvelius, G., Nordling, K., Jörnvall, H., Bergman, T., Johansson, J. (2009) Peptide-binding specificity of the prosurfactant protein C Brichos domain analyzed by electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom. 23: 3591-8.
https://doi.org/10.1002/rcm.4282

VIII. Willander, H., Askarieh, G., Landreh, M., Westermark, P., Nordling, K., Keränen, H., Hermansson, E., Hamvas, A., Nogee, L.M., Bergman, T., Saenz, A., Casals, C., Åqvist, J., Jörnvall, H., Berglund, H., Presto, J., Knight, S.D., Johansson, J. (2012) High-resolution structure of an intramolecular chaperone. Implications for anti-amyloid activity of the BRICHOS domain. Proc Natl Acad Sci USA. 109: 2325-9.
https://doi.org/10.1073/pnas.1114740109

IX. Peng, S., Fitzen, M., Jörnvall, H., Johansson, J. (2010). The extracellular domain of Bri2 (ITM2B) binds the ABri peptide (1-23) and amyloid beta-peptide (Abeta1-40). Implications for Bri2 effects on processing of amyloid precursor protein and Abeta aggregation. Biochem Biophys Res Commun. 393: 356-61.
https://doi.org/10.1016/j.bbrc.2009.12.122

X. Landreh, M., Askarieh, G., Nordling, K., Hedhammar, M., Rising, A., Astorga-Wells, J., Alvelius, G., Casals, C., Knight, S. D., Johansson, J., Jörnvall, H., and Bergman, T. (2010) A pH-dependent dimer lock in spider silk protein. J Mol Biol. 404: 328-36.
https://doi.org/10.1016/j.jmb.2010.09.054

XI. Jaudzems, K., Askarieh, G., Landreh, M., Nordling, K., Hedhammar, M., Jörnvall, H., Rising, A., Knight, S. D., and Johansson, J. (2012) pH-dependent dimerization of spider silk N-terminal domain requires relocation of a wedged tryptophan side-chain. J Mol Biol. 422: 477-87.
https://doi.org/10.1016/j.jmb.2012.06.004

XII. Rising, A., Nordling, K, Landreh, M., Lindqvist, A., Johansson, J. The N-terminal domain of spider silk proteins as solubility-enhancing tag for recombinant protein production. [Manuscript]

XIII. Landreh, M., Johansson, J., Rising, A., Presto, J., and Jörnvall, H. (2012) Control of amyloid assembly by autoregulation. Biochem J. 447: 185-92.
https://doi.org/10.1042/BJ20120919