Research

July 2004

July 22, 2004

Medical Relevance of Amyloid aggegation.

Posted by David Talaga

Aggregation of soluble polypeptides or proteins into insoluble amyloid fibrils containing the cross-ß structural motif has been observed in the progression of a great variety of diseases. There are over 20 diseases1 that have been linked to excessive deposits of amyloid fibrils or plaques derived from different precursor proteins. Amyloidogenic diseases include Alzheimer’s disease, Parkinson’s disease, type II diabetes, and spongiform encephalopathies. The human health impact of these diseases has motivated intensive study and numerous reviews of the structure and growth of amyloid fibrils.1-19

A mechanistic understanding of the amyloid-assembly process will provide new handles and probes for the physiological interactions that cause amyloidosis. This will allow better approaches to the prevention of amyloid formation and new diagnostics for early detection of amyloid-related diseases. A popular hypothesis is that blocking and/or reversing amyloid formation will be an effective treatment for diseases involving organ failure due to amyloidosis. To adequately test this hypothesis, rational strategies must be based on interrupting or reversing amyloid aggregation at various points in amyloid assembly. This requires detailed knowledge of the mechanisms of amyloid growth and the factors that influence the (dis)aggregation rates at all stages of amyloid assembly.20

Of particular interest is the participation of the difficult-to-detect-and-quantify species present during the lag phase of amyloid assembly. Recent evidence has shifted some of the focus from amyloid fibrils to prefibrillar amyloidogenic aggregates as the cause of Alzheimer’s disease symptoms.2 Development of vaccines targeting small amyloidogenic aggregates 6,7,12,13,21 would benefit from understanding how the concentrations of amyloidogenic species will be influenced by the induced immunological response to and clearing of particular amyloid species. A species that is too small to seed further amyloid assembly could, in principle, be “deactivated” if it is known what structural part of the species is required for further aggregation to the critical size for nucleation of amyloid fibrils. This structural part could be tested as a vaccine to induce immunological clearing of a particular amyoidogenic species with a lower likelihood of inducing further amyloid plaque deposition from the introduction of an actual amyloidogenic species.

(1) Sipe, J. D.; Cohen, A. S. "Review: History of the amyloid fibril" Journal of Structural Biology 2000, 130, 88.

(2) Walsh, D. M.; Hartley, D. M.; Selkoe, D. J. "The many faces of Aß: Structures and activity" Current Medicinal Chemistry: Immunology, Endocrine & Metabolic Agents 2003, 3, 277.

(3) Temussi, P. A.; Masino, L.; Pastore, A. "From alzheimer to huntington: Why is a structural understanding so difficult?" EMBO Journal 2003, 22, 355.

(4) Buxbaum, J. N. "The systemic amyloidoses" Current Opinion in Rheumatology 2003, 16, 67.

(5) Wetzel, R. "Ideas of order for amyloid fibril structure" Structure (Cambridge, MA, USA) 2002, 10, 1031.

(6) Solomon, B. "Towards alzheimer's disease vaccination" Mini-Reviews in Medicinal Chemistry 2002, 2, 85.

(7) Schenk, D. "Opinion: Amyloid-ß immunotherapy for alzheimer's disease: The end of the beginning" Nature Reviews Neuroscience 2002, 3, 824.

(8) Murphy, R. M. "Peptide aggregation in neurodegenerative disease" Annual Review of Biomedical Engineering 2002, 4, 155.

(9) Makin, O. S.; Serpell, L. C. "Examining the structure of the mature amyloid fibril" Biochemical Society Transactions 2002, 30, 521.

(10) Kirkitadze, M. D.; Bitan, G.; Teplow, D. B. "Paradigm shifts in alzheimer's disease and other neurodegenerative disorders: The emerging role of oligomeric assemblies" Journal of Neuroscience Research 2002, 69, 567.

(11) Holtzman, D. M. "Aß conformational changes is central to alzheimer's disease" Neurobiology of Aging 2002, 23, 1085.

(12) Hardy, J.; Selkoe, D. J. "The amyloid hypothesis of alzheimer's disease: Progress and problems on the road to therapeutics" Science (Washington, DC, USA) 2002, 297, 353.

(13) Talaga, P. "Beta-amyloid aggregation inhibitors for the treatment of alzheimer's disease: Dream or reality?" Mini Rev Med Chem 2001, 1, 175.

(14) Dobson, C. M. "Protein misfolding, evolution and disease" Trends in Biochemical Sciences 1999, 24, 329.

(15) Teplow, D. B. "Structural and kinetic features of amyloid ß-protein fibrillogenesis" Amyloid 1998, 5, 121.

(16) Fink, A. L. "Protein aggregation: Folding aggregates, inclusion bodies and amyloid" Folding & Design 1998, 3, R9.

(17) Rochet, J.-C.; Lansbury, P. T., Jr. "Amyloid fibrillogenesis: Themes and variations" Current Opinion in Structural Biology 2000, 10, 60.

(18) Serpell, L. C. "Alzheimer's amyloid fibrils: Structure and assembly" Biochimica et Biophysica Acta 2000, 1502, 16.

(19) Kad, N. M.; Radford, S. E. "Partial unfolding as a precursor to amyloidosis: A discussion of the occurrence, role, and implications" Frontiers in Molecular Biology 2001, 37, 257.

(20) Dominguez, D. I.; De Strooper, B. "Novel therapeutic strategies provide the real test for the amyloid hypothesis of alzheimer's disease" Trends in Pharmacological Sciences 2002, 23, 324.

(21) Bruce-Keller, A. J.; Estus, S. "Concern over the amyloid vaccine: Amyloid heterogeneity and fc receptor signaling" Neurobiology of Aging 2002, 23, 667.

July 7, 2004

Amyloid Assembly Background

Posted by David Talaga

Fibril 4A

Medical Relevance
Aggregation of soluble polypeptides or proteins into insoluble amyloid fibrils with cross-ß structural motif has been observed in the progression of a great variety of diseases. Over 20 diseases1 have been linked to excessive deposits of amyloid fibrils or plaques derived from different precursor proteins. Amyloidogenic diseases include Alzheimer’s disease, Parkinson’s disease, type II diabetes, and spongiform encephalopathies. The human health impact of these diseases has motivated intensive study and numerous reviews of the structure and growth of amyloid fibrils.1-19
A mechanistic understanding of the amyloid-assembly process will provide new handles and probes for the physiological interactions that cause amyloidosis. This will allow better approaches to the prevention of amyloid formation and new diagnostics for early detection of amyloid-related diseases. A popular hypothesis is that blocking and/or reversing amyloid formation will be an effective treatment for diseases involving organ failure due to amyloidosis. To adequately test this hypothesis, rational strategies must be based on interrupting or reversing amyloid aggregation at various points in amyloid assembly. This requires detailed knowledge of the mechanisms of amyloid growth and the factors that influence the (dis)aggregation rates at all stages of amyloid assembly.20
Of particular interest is the participation of the difficult-to-detect-and-quantify species present during the lag phase of amyloid assembly. Recent evidence has shifted some of the focus from amyloid fibrils to these prefibrillar amyloidogenic aggregates as the cause of Alzheimer’s disease symptoms.2 Development of vaccines targeting small amyloidogenic aggregates 6,7,12,13,21 would benefit from understanding how the concentrations of amyloidogenic species will be influenced by the induced immunological response to and clearing of particular amyloid species. A species that is too small to seed further amyloid assembly could, in principle, be “deactivated” if it is known what structural part of these species is required for further aggregation to the critical size for nucleation of amyloid fibrils. This structural part could be tested as a chimeric vaccine to induce immunological clearing of that particular amyoidogenic species with a lower likelihood of inducing further amyloid plaque deposition from the introduction of an actual amyloidogenic species.

Structural Features of Amyloid
Amyloid deposits derived from diverse precursors share many structural features.
Core structure:  Experiments suggest that amyloid fibrils share a common core filament structure, irrespective of the nature of their precursor proteins. X-ray22  and electron diffraction9,23-27 studies of amyloid fibrils have confirmed the generality of the cross-ß helical structure present in amyloid with ß-Strands separated by 4.7Å18 and ß-sheets separated by 9.8Å. The ß-sheet structure has the strands perpendicular to the long axis of the fibril and hydrogen bonded along the axis of the fibril.22,28-31 Filaments typically have two or more ß-sheets that are stacked normal to the helical axis and extend along it. Fibrils are composed of two or more filaments.25,32-35  Histological staining of various amyloid deposits exhibits a common behavior. Congo red shows green birefringence and thioflavin T (ThT) shows a new profluorescent absorption band36-38 that that are both thought to be related to the common cross-ß core structure. Recently, linear birefringence and dichroism of Congo red has been used to determine the relative orientation of fibrils within amyloid plaques in situ.39 This type of non-covalent labeling is sensitive to the presence of the quaternary interactions specific to fully-formed amyloid.
NMR has also been used to determine the residue-level participation in the core structure of amyloid fibrils using H/D exchange40-44 and relaxation measurements.45-50 These results show that the segments of ß-stands that comprise the core of the fibrils is are protected from access to water and are more rigidly held than the loops or ends that are not part of the core.22

Conformational changes are typically observed during amyloid assembly. In their native state, the precursor proteins may not, in general, contain the secondary structural elements present in the final amyloid assembly. The amide I infrared absorption or Raman band is typically observed to lose intensity associated with the native state and gain intensity associated with cross-ß.51-59 Circular dichroism of the peptide backbone absorption band is also sensitive to secondary structure and gives similar results.23,60-63 Fluorescence spectroscopy can also be used to detect conformational changes either by non-covalent labeling with dyes like ANS that are specific for exposed hydrophobic patches64-70 or through covalent attachment of fluorescent dyes.71-74 Infrared absorption and Raman suffer from solvent interferences and are often performed on powders. We use circular dichroism to confirm the correlation of covalent and non-covalent fluorescent labeling schemes with secondary structural changes.