Research Results - Amyloid

Amyloid aggregation mechanisms

The study of amyloid structure and growth has been motivated by their implication in many human diseases. There are ~20 diseases associated with excessive deposits of amyloid plaques in the affected tissue or organ including Alzheimer?s disease (AD), Parkinson?s disease (PD), type II diabetes, and spongiform encephalopathies. In these disease states, proteins that are normally soluble undergo aggregation to form various intermediates and amyloidogenic species. These species subsequently assemble to generate insoluble fibrils that accumulate in the affected tissues or organs. A detailed understanding of amyloid growth mechanisms will allow new approaches to the prevention of amyloid formation and better diagnostics for early detection of amyloidogenic diseases.

A molecular-level mechanism of how the different amyloid species interconvert is the goal of this project. There are many species of amyloid particles present physiologically. Our single molecule studies aim to classify the species involved in amyloid formation according to size, shape, kinetic reactivity, and monomer 2° and 3&degree; structural information. A molecular-level mechanism of amyloid growth must include details as to when the protein misfold occurs and how it is influenced by the dynamics of protein structure. To determine the physical interactions and structural changes involved in the amyloid assembly mechanism, we study effect of environmental variables such as temperature, pH, helix promoting solvents, denaturants, and reducing agents. The environmental effect on aggregation is expected to be species-dependent reflecting a possible hierarchy of structural interactions.

During their life cycle proteins undergo many types ofconformational changes. After folding, the activity and regulation of proteins are also mediated by conformational changes. Misfolded and damaged proteins are usually degraded. However, irreversible aggregation into amyloid is accompanied by yet another conformational change.

Protein Conformational Dynamics Background

During their life cycle proteins undergo many types of conformational changes.  The first type of conformational change is the process of protein folding.  The polypeptide must fold into its active three-dimensional structure before it is degraded by cellular proteases or aggregates. In fact this process is so important that organisms have developed systems such as Chaperonins that consume energy to assist in the folding process. Once the protein has obtained its proper three-dimensional structure it may undergo further structural changes that are associated with its function. Many enzymes and ligand-binding proteins must undergo large conformational changes during their function. The activity of a protein can be modulated by allosteric interactions with other molecules. These interactions result in a dynamic structural change that changes the protein's activity. Finally, most proteins only survive a short time in the cell without becoming damaged.  Damage often will change the structure of the protein and allow proteases to degrade it allowing the cycle to begin anew.  If the degradation process is faulty then aggregation can occur.  Thus, during their life cycle proteins can undergo five basic classes of conformational change.

  • Folding
  • Activity
  • Regulation
  • Degradation
  • Aggregation
Defects in these processes can result in diseases.

Fibril 4A

Amyloid Assembly Background

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.

Medical Relevance of Amyloid aggegation.

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.

Overview of amyloid Assembly mechanisms

Overview of amyloid assembly mechanisms

Amyloid – any fibril, plaque, seed, or aggregate that has the characteristic cross-ß sheet structure.
Amyloidogenic precursor – a protein or peptide that upon incubation under appropriate conditions will form amyloid fibrils or plaques.
Amyloid fibril – long ribbons of amyloid ~10nm in diameter and >100nm in length. Most often observed in vitro.
Amyloid plaque – the form of amyloid most often found in vivo – often comprised of aggregated amyloid fibrils.
Amyloid protofibril/filament – a species of amyloid smaller in diameter (3-6nm) and length(<100nm) than typical for amyloid fibrils, thought to be a possible direct precursor to amyloid fibrils perhaps through lateral aggregation.
Amyloid seed (or template) – a species of a critical size or structure that rapidly elongates to form larger amyloid species possibly by providing a proper scaffold for amyloid assembly
Amyloidogenic oligomer – A small aggregate of precursor that is smaller than the critical “seed” size but still may have some of the structural characteristics of amyloid.
Amyloidogenic fold – a structure of the precursor that must be accessed prior to amyloidogenic aggregation, thought to retain substantial secondary structure possibly including some of the native fold. It could be related to a misfolded or molten globule structure.
Folded state – The native (functional) state of the precursor.
Folding intermediate – A partially folded or misfolded structure of the precursor. These partially folded structures are potentially the same as or precursors to amyloidogenic folds.
Denatured state – The unfolded state of the precursor.
Unstructured aggregate – Completely or partially denatured proteins tend to aggregate non-specifically without forming a particular structural motif

Assembly Image Steps.png

Chromophore counting by discrete photobleaching events.

200610191240

Fibril flow through1.jpg

Development of micro/nano fluidic devices for Amyloid Detection

June 22, 2005

Diffusion Hmm-1

Hidden Markov Models for Diffusive Processes

The top panel (a) shows a Monte Carlo simulated trajectory of a molecule diffusing through a spherically symmetric Gaussian collection volume. The next panel (b) shows the reciprocal of the inter-photon time. The delay between excitation and emission of the photon is the lifetime and is shown in panel (c). Panels (b) and (c) are used with three dimensional diffusion of the molecule through the focus of the instrument as the hidden Markov model to reconstruct the Brownian motion trajectory in panel (d). The trajectory was reconstructed by using Monte Carlo sampling of the HMM parameters to maximize the likelihood of the data in (b) and (c).

Afm28 10Um 1-3

Beta-lactoglobulin aggregation visualized by AFM

This is an image of ß-Amyloid fibrils derived from ß-lactoglobulin taken using tapping-mode AFM in air on a piece of mica that has been chemically modified to bind our sample. Note the great diversity of size, shape, and height of the species present. Many different types of aggregates are visible in this image including filaments, fibrils, and small aggregates of various sizes.

Messina2006

Hidden Markov Model Analysis of Multichromophore Photobleaching

The interpretation of single-molecule measurements is greatly complicated by the presence of multiple fluorescent labels. However, many molecular systems of interest consist of multiple interacting components. We address this issue using multiply-labeled dextran polymers that we intentionally photobleach to the background on a single molecule basis. Hidden Markov models allow unsupervised analysis of the data to determine the number of fluorescent subunits involved in the fluorescence intermittency of the 6-carboxy-tetramethylrhodamine labels by counting the discrete steps in fluorescence intensity. The Bayes information criterion allows us to distinguish between hidden Markov models that differ by number of states, i.e., number of fluorescent molecules. We determine information-theoretical limits and show via Monte Carlo simulations that the hidden Markov model analysis approaches these theoretical limits. This technique has resolving power of one fluorescing unit up to as many as 30 fluorescent dyes with the appropriate choice of dye and adequate detection capability. We discuss the general utility of this method for determining aggregation-state distributions as could appear in many biologically important systems and its adaptability to general photometric experiments.

Nanopore Amyloid

Solid-state nanopore measurements of amyloid formation

In collaboration with the group of Jiali Li at the University of Arkansas Department of Physics we have begun making solid state nanopore electrical measurements on the aggregates that form during assembly of amyloid from ß-lactoglobulin. Our preliminary assessment of the data suggests that the solid state nanopore can distinguish between different classes of amyloid species.

β-Lactoglobulin Calyx Dynamics

β-Lactoglobulin Calyx Dynamics
The formation of a hydrophobic core provides a large part of the driving force for protein folding.
We are investigating amyloid formation from β-lactoglobulin and are therefore interested in understanding how the dynamics of core stability are influenced by solution conditions.
We have exploited the presence of a large hydrophobic binding pocket in β-lactoglobulin to encapsulate coumarin 153 (C153).
Steady state spectroscopy reveals a very blue-shifted spectrum consistent with an environment similar to a combination of hexane and toluene.
Time resolved fluorescence Stokes shift measurements reflect the dynamics of the hydrophobic core of β-lactoglobulin.
A transition in the dynamics of the hydrophobic core occurs at a lower temperature than does the melting of the protein as measured by circular dichroism suggesting a partitioning of  the enthalpy and entropy balance between the core structure and secondary structure.