Section: Protein aggregation
We are interested in following how different proteins can form well-ordered fibrils. We study the following proteins:
(A) Pathological aggregation:
α-synuclein and Parkinson's Disease:
α-synuclein (αSN) is the major protein involved in the formation of insoluble deposits known as Lewy Bodies in the brains of Parkinson's patients. αSN is natively unfolded in solution, but assumes amyloid structures spontaneously. We conduct basic research on how α-synuclein aggregates (1, 2) and forms non-amyloid structures in lipids (3). We recently solved a low-resolution structure of what is thought to be the oligomeric intermediate in the aggregation process (2) (see picture below) and are continuing to study the properties of this oligomer. In addition, we are involved in a large project with Pfizer to develop compounds that inhibit the aggregation of αSN with the aim of developing drugs against the disease. We have developed assays (4, 5) that have allowed us with Pfizer to complete a high-throughput screen of compounds that inhibit αSN aggregation and are now analyzing the best of these compounds for their molecular properties.
We have also shown that αSN aggregates in a fundamentally different manner in the presence of SDS,
forming what looks like beads on a string (1) - as shown in the figure below. Here there is a (light gray) shell of protein surrounding the (dark gray) hydrocarbon core of the SDS micelles, and each bead is presumably linked by amyloid-forming bridging segments.
Fas4 and corneal dystrophies:
Several corneal dystrophies (CD) arise from the deposition of protein as aggregates in the otherwise transparent corneal tissue, ultimately leading to blindness. Transforming growth factor Beta Induced protein (TGFBIp) is the major component in many of these diseases, and we are studying the mechanism by which this aggregation occurs in order to develop ways to prevent it. TGFBIp is a 683-residue protein consisting of four domains. We have shown that the C-terminal domain (Fas4) is a good model for the behavior of the entire protein in terms of how CD-inducing mutants affect stability of the protein (6). Interestingly, different mutations in Fas4 lead to different aggregates, some of which are amyloid-like and others of which are more amorphous. We are continuing these studies with Fas4 to understand the molecular mechanisms behind these unwanted processes.
(B) Functional aggregation
Functional or useful amyloid illustrates how Nature makes good use of something which is potentially dangerous. When amyloid does occur in Nature as a beneficial phenomenon (e.g. in melanosomes, insect cocoons and bacterial biofilm), it is because the amyloid-producing organisms have developed strategies to control amyloid formation in time and space.
We have shown that the main amyloid protein in E. coli called CsgA, which produces curli, fibrillates under a wide variety of conditions - i.e. it is really "hardwired" to fibrillate (7). We have developed assays to identify the occurrence of amyloid structures in different bacterial communities, and have shown that this very wide-spread, with up to 50% of all bacterial species producing amyloid (8, 9), including several pathogenic Gram-positive species (10). We recently identified a new amyloid-producing operon in the widespread Pseudomonas family (11).
This operon is organized in a different way to E. coli's curli operon and we are currently analyzing the different components involved in this. Our work has been summarized in several recent reviews (12, 13).
(C) Model systems of aggregation:
Glucagon: This 29-residue peptide hormone does not fibrillate under physiological conditions, but has a high tendency to do so during production in the pharmaceutical industry. We have carried out numerous studies of how different types of fibrils are formed under different circumstances, illustrating the principle of fibrillar polymorphism (14-25). This has been summarized in several reviews (26, 27).
S6 (ribosomal protein from T. thermophilus): With the study of fibrillation of S6 at low pH, we were the first group to publish a detailed protein engineering study of a protein that can be induced to fibrillate with a pronounced lag time from a quasi-native state, demonstrating that fibrillation requires partially unfolded regions and that "minimization" of the backbone by truncation to alanine favours this process (28).
Protein detergent interactions
- 1. Giehm, L., Oliveira, C. L. P., Christiansen, G., Pedersen, J. S., and Otzen, D. E. (2010) SDS-induced fibrillation of α-synuclein: An alternative fibrillation pathway, Journal of Molecular Biology 401, 115-133.
- 2. Giehm, L., Svergun, D. I., Otzen, D. E., and Vestergaard, B. (2011) Low resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation, Proc. Natl. Acad. Sci. U.S.A. 108, 3246-3251.
- 3. Kjær, L., Giehm, L., Heimburg, T., and Otzen, D. E. (2009) The influence of vesicle composition and size on -synuclein structure and stability, Biophys. J. 96, 2857-2870.
- 4. Giehm, L., Lorenzen, N., and Otzen, D. E. (2010) Assays for α-synuclein aggregation, In Methods (Cyr, D., Ed.), Elsevier.
- 5. Giehm, L., and Otzen, D. E. (2010) Strategies to increase the reproducibility of α-synuclein fibrillation in plate reader assays, Anal. Biochem. 400, 270-281.
- 6. Runager, K., Basaiawmoit, R. V., Deva, T., Andreasen, M., Valnickova, Z., Sørensen, C. S., Karring, H., Thøgersen, I. B., Christiansen, G., Underhaug, J., Kristensen, T., Nielsen, N. C., Klintworth, G. K., Otzen, D. E., and Enghild, J. J. (2011) Human phenotypically distinct TGFI corneal dystrophies are linked to the stability of the fourth Fas1 domain of TGFBIp, J. Biol. Chem. 286, 4951-4958.
- 7. Dueholm, M., Nielsen, S. B., Hein, K. L., Nissen, P., Chapman, M. R., Christiansen, G., Nielsen, P. H., and Otzen, D. E. (2011) Fibrillation of the Major Curli Subunit CsgA under changing conditions implies robust design of aggregation, Biochemistry In press.
- 8. Larsen, P., Dueholm, M., Christiansen, G., Nielsen, J. L., Otzen, D. E., and Nielsen, P. H. (2007) Amyloid adhesins are abundant in natural biofilms, Env. Microbiol. 9, 3077-3090.
- 9. Larsen, P., Nielsen, J. L., Otzen, D. E., and Nielsen, P. H. (2008) Amyloid-like adhesins in floc-forming and filamentous bacteria in activated sludge, Appl. Env. Microbiol. 74, 1517-1526.
- 10. Jordal, P. B., Dueholm, M., Larsen, P., Pedersen, S. V., Enghild, J. J., Christiansen, G., Højrup, P., Nielsen, P. H., and Otzen, D. E. (2009) Widespread abundance of Functional Bacterial Amyloid in Mycolata and other Gram positive Bacteria, Appl. Env. Microbiol. 75 4101-4110.
- 11. Dueholm, M. S., Petersen, S. V., Sønderkær, M., Larsen, P., Christiansen, G., Hein, K. L., Enghild, J. J., Nielsen, J. L., Nielsen, K. L., Halkjær, P. H., and Otzen, D. E. (2010) Functional Amyloid in Pseudomonas, Mol. Microbiol. 77, 1009-1020.
- 12. Nielsen, P. H., Dueholm, M. S., Thomsen, T. R., Nielsen, J. L., and Otzen, D. E. (2010) Functional bacterial amyloids in biofilms, In Annual Biofilm Highlights (Flemming, H.-C., Szwezyk, U., and Wingender, J., Eds.).
- 13. Otzen, D. E., and Nielsen, P. H. (2008) We find them here, we find them there: Functional bacterial amyloid, Cell. Mol. Life Sci. 65, 910-927.
- 14. Dong, M., Hovgaard, M. B., Xu, S., Otzen, D. E., and Besenbacher, F. (2006) AFM study of glucagon fibrillation via oligomeric structures resulting in interwoven fibrils, Nanotechnology 17, 1-6.
- 15. Pedersen, J. S., Dikov, D., Flink, J. L., Hjuler, H. A., Christiansen, G., and Otzen, D. E. (2006) The changing face of glucagon fibrillation: Structural polymorphism and conformational imprinting, Journal of Molecular Biology 355, 501-523.
- 16. Pedersen, J. S., Dikov, D., Flink, J. L., and Otzen, D. E. (2006) Sulfates dramatically stabilize a salt dependent type of glucagon fibrils, Biophys. J. 90, 4181-4194.
- 17. Pedersen, J. S., Dikov, D., and Otzen, D. E. (2006) N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon, Biochemistry 45, 14503-14512.
- 18. Andersen, C. B., Otzen, D. E., Christiansen, G., and Rischel, C. (2007) Glucagon amyloid-like fibril morphology is selected via morphology-dependent growth inhibition, Biochemistry 46, 7314-7324.
- 19. Hovgaard, M. B., Dong, M., Otzen, D. E., and Besenbacher, F. (2007) Quartz Crystal Microbalance studies of multi-layer glucagon fibrillation at the solid-liquid interface., Biophys. J. 93, 2162-2169.
- 20. Christensen, P. A., Pedersen, J. S., Christiansen, G., and Otzen, D. E. (2008) Spectroscopic Evidence for the Existence of an Obligate Pre-Fibrillar Oligomer during glucagon fibrillation, FEBS Lett. 582, 1341-1345.
- 21. Dong, M., Hovgaard, M. B., Mamdouh, W., Xu, S., Otzen, D. E., and Besenbacher, F. (2008) AFM-based force spectroscopy measurements on mature amyloid fibrils of the peptide glucagon, Nanotechnology 19, 384013 (384017pp).
- 22. Svane, A. S. P., Jahn, K., Deva, T., Malmendal, A., Otzen, D. E., Dittmer, J., and Nielsen, N. C. (2008) Early Stages of Amyloid Fibril Formation Studied by Liquid-State NMR: The Peptide Hormone Glucagon, Biophys. J. 95, 366-377.
- 23. Oliveira, C. L. P., Behrens, M. A., Pedersen, J. S., Erlacher, K., Otzen, D. E., and Pedersen, J. S. (2009) SAXS Study of Glucagon Fibrillation: From Monomers to Fibers, Journal of Molecular Biology 387, 147-161.
- 24. Andersen, C. B., Hicks, M. R., Vandahl, B., Rahbek-Nielsen, H., Thøgersen, H., Thøgersen, I. B., Enghild, J. J., Serpell, L. C., Rischel, C., and Otzen, D. E. (2010) Glucagon fibril polymorphism reflects differences in protofilament backbone structure, Journal of Molecular Biology 397, 932-946.
- 25. Macchi, F., Hoffmann, S. V., Carlsen, M., Vad, B., Imparato, A., Rischel, C., and Otzen, D. E. (2011) Mechanical stress affects glucagon fibrillation kinetics and fibril structure, Langmuir In press.
- 26. Pedersen, J. S., Andersen, C. B., and Otzen, D. E. (2010) Institutional nonconformism: the many levels of glucagon polymorphism, FEBS J. 277, 4591-4601.
- 27. Pedersen, J. S., and Otzen, D. E. (2008) Amyloid - A state in many guises: survival of the fittest fibril fold, Protein Science 17, 1-9.
- 28. Pedersen, J. S., Christiansen, G., and Otzen, D. E. (2004) Modulation of S6 fibrillation by unfolding rates and gatekeeper residues, Journal of Molecular Biology 341, 575-588.