Amyloid formation, a protein dysfunction leading to fibril plaques in vital organs, is a source of several debilitating diseases. In this paper I shall first explore the implications of amyloid formation. I will then explicate proposed models for the structure of amyloid and conditions leading to its formation. I will show how amyloid is implicated in Alzheimer's disease, concentrating on the Amyloid Precursor Protein (APP) which precedes it, and also on other proteins suspected to be involved in this complex disease. I will explain several proposed models for inhibiting or slowing the disease process with this knowledge, and then explain current research in the field. Finally, I shall elucidate my own proposed experiments for determining the function and integration of b-APP in Alzheimer's amyloid formation.
Note: b = beta
Protein misfolding resulting in malfunction of cellular machinery causes a broad range of debilitating diseases (1). Amyloid, one type of misfolding event, occurs when proteins or protein fragments unfold from their soluble forms to form insoluble fibrils. Frequently these fibrils take the form of a b-sheet, hence the term b-amyloid. These fibrils accumulate in a variety of organs (i.e. the spleen, pancreas, and brain) to cause diseases such as Alzheimer's, Type II Diabetes, and Cretuzfeldt-Jakob disease.(1)
A wide range of proteins with unique folds (i.e. lysozyme and transthyritin) dismantles to form similarly structured b-amyloid plaques. Furthermore, evidence has shown that proteins not known to form amyloid under natural conditions may do so under denaturing conditions (i.e., in the presence of urea).1 Proposed models for amyloid show stabilizing molecular bonds formed by the peptide backbone holding the structure together, which explains the susceptibility of any protein to amyloid formation.
Under physiological conditions, amyloid fibrils are essentially indestructible. It has been proposed that evolution has avoided this structure by selecting sequences which efficiently fold into globular form to hide the polypeptide chain and hydrophobic residues from the hydrophilic exterior. Another hypothesis involves the rate at which proteins fold: a rate so fast it is near to the limits imposed on all chemical reactions. This rate of folding would minimize time spent in an unfolded state, thus competing out the intermolecular processes of aggregation and yielding less a chance of amyloid formation.
Despite evolutionary avoidance of amyloid formation, it still occurs and causes severely debilitating diseases. Once formed, amyloid fibrils are nearly indestructible and controlling their growth is nearly impossible. One has only to consider the effect of Alzheimer's (an amyloid-related malfunction resulting in deterioration and malfunction of the brain) on former president Ronald Reagan to realize the continued importance of amyloid study.
Formation and Structure
Several factors lead to the formation of amyloid. In vitro, conditions leading to denaturation (low pH, urea presence) have been shown to induce amyloid. In vivo, one possible mechanism has been proposed (Figure 1). After synthesis, the protein is folded on the ER with the help of molecular chaperones (which deter aggregation). It is then transported out of the cell to provide its normal function in the extracellular environment. Certain conditions induce the native protein to denature (at least partially), and it is this partially-denatured intermediate between the folded (N) and the unfolded (U) forms that is susceptible to aggregation. The aggregated denatured proteins form insoluble plaques, which form deposits in organs such as the brain or eye.(1)
Figure 1. Proposed model for amyloid formation.
The following image shows a proposed model of amyloid formation. In this model, the protein leaves the cell and in the extracellular space encounters denaturing conditions. It subsequently unfolds (or partially unfolds) and becomes susceptible to amyloid fiber formation. Taken from TiBS, September 1999.
Studies have shown that protein peptides convert to amyloid fibers by forming 'cross-b' structures with H-bonds between chains, oriented in manner parallel to the fiber axis itself (Figure 2). These b chains wind around in a helical manner, creating a hydrophobic interior and hydrophilic exterior. One model formed from the protein transthyretin shows a repeating unit of 24b strands, which form a complete helical turn of b sheet about an axis parallel to the fiber axis. This simplistic, though incredibly stable structure is what makes amyloid fibrils nearly indestructible.
Figure 2. Amyloid structure.
The following show one proposed structure of amyloid fibers, a) explicates the cross-b sheets running antiparallel to the fiber axis, b) shows the cross-b sheets coming together in helical form to make a fiber.
a) Taken from Intro. to Protein Structure
b) Taken from TiBS, September 1999.
Alzheimer's : Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposit of fibrillar proteins within the brain as amyloid plaques, causing an atrophy of grey matter composed of dead neurons and synapses. Several genetic precursors have been implicated in AD-related amyloid formation. One mutation causally linked to familial Alzheimer's occurs in what is known as the b-amyloid precursor protein (APP) gene on chromosome 21. The major component of the fibril plaques is the amyloid-b (Ab) peptide, which is a member of the APP family.(3) It has been hypothesized that Ab is formed by the action of secretases on the APP precursor, in a manner shown in Figure 3.
Figure 3. APP cleavage
to form amyloidogenic Ab peptide.
APP is thought to be a precursor to Ab peptide, a precursor to amyloid formation.
APP is processed through secretory pathways of neurons and other cells which subsequently secrete Ab peptides 40- and 42-residues long (Ab1-40 and Ab1-42). These peptides are identical to those identified in neuritic plaques, and APP most likely is part of an undetermined chain of protein interactions which lead to the secretion of amyloid-related peptides. In vitro it has been shown that oligomeric Ab1-40 and Ab1-42 are neurotoxic, further cementing the claim that these peptides secreted in the presence of APP lead to amyloidogenic diseases such as Alzheimer's. (4)
Solutions so far
Several solutions have been proposed at this level of complexity (or lack thereof). These are explicated in Figure 4. The first strategy is simply repression of the APP gene. Down Syndrome people are shown to have an over-expression of this gene, and a correlating early occurrence of Alzheimer's. Thus, it is believed that repression will minimize the effects of the gene.(4) As discussed below, however, the APP protein is believed to have some unknown role in learning mechanisms.5 Furthermore, suppression of the gene in mice has shown severe neurological disorders, ultimately suggesting that quelling the gene in human subjects would be unwise.6 Another strategy involves inhibition of the expression of Ab1-42 and/or Ab1-40, as these peptides are found in nearly phenotypically-identical subjects for Alzheimer's. Ab1 is, however, only a small part of total Ab secretion, and thus may be functionally insignificant.(5)
Further strategies therapeutic intervention at the fibrillization level, either by inhibiting early intermediates of Ab formation (via apoE, which inhibits nucleation of fibrils) or by blocking the neurodegenerative effects of Ab fibrils.(5)
Figure 4. Proposed methods of therapeutic intervention in Alzheimer's-implicated Ab peptide formation.(5)
The following shows a schematic for suggested interventions between amyloid proteins and factors known to be central to their formation in AD.
One Further Piece of the Puzzle: Internalization of
APP by the X11 PTB Domain
The X11 protein, function unknown, has been found to bind to the a domain of the b-amyloid precursor protein (APP). This complex has been isolated (Figure 5). The PTB domain on the X11 protein is a phosphotyrosine-binding/ phosphotyrosine interaction domain. Peptide residues that are N-terminal to the phosphotyrosine interact with the PTB domain. A b-turn formed by the motif is critical for recognition.
X11, a neuron-specific protein,
has been found to bind via its PTB domain to a specific domain
of the b-APP in vivo. The biological consequence of this
complex is unknown but it has been determined that the binding
occurs both with high affinity and high specificity, suggesting
Figure 5. Internalization
motif of Alzheimer's APP complexed with PTB domain of X11 protein.
The following is a Rasmol image of the internalization sequence from the amyloid precursor protein of Alzheimer's with the phosphotyrosine-binding/phosphotyrosine interacting domain (PDB) of protein X11.
A 14-residue peptide portion
of the amyloid precursor protein is found to compete with the
full APP for the PTB domain of X11, suggesting that the particular
sequence (QNGEYNPTYKFFEQ) is accountable for internalization
of b-APP by X11. This internalization of b-APP leads to the formation
of pathological b-amyloid peptides. Elimination of the X11 PTB
binding site on b amyloid precursor protein diminished its internalization
in X11 and also decreased the production of amyloidogenic Ab peptide.(6)
Little official knowledge is
currently held about the structures and roles of either b-APP
or X11. b-APP is thought to have an important role in learning,
though this has yet to be elucidated.(6) As previously stated,
X11 has no known function, but its high affinity and specificity
for the 14-residue portion of b-APP, as well as the effects of
eliminating the PTB binding site on b-APP, lead one to believe
that it is of some biological importance and is certainly critically
relevant in amyloid-implicated Alzheimer's disease.
The implication of X11 in the conversion of b-APP to b-amyloid peptides is huge. Further study of the role of this protein in physiological processes is key, and could be done by altering the protein in mice and monitoring effects. The role that APP is thought to have in learning may be affected by altering its PTB binding site and so it may be worthy to consider making changes in X11 instead. It is possible that altering the active site of X11 by one or two residues could seriously affect its ability to bind b-APP and form amyloid fibers, and this might be done via point mutation in base pairs at the level of RNA.
Extensive study of both APP and other proteins involved in its amyloidic effect seems a likely route of uncovering a cure for Alzheimer's disease. Most of these studies would have to be done posthumously, which is a limitation to distinguishing APP's exact route to amyloid formation. It would be worthy to give significant attention to the structure of X11, and especially it's PTB site where it concerns binding with b-APP. Utilizing amide proton exchange may be a useful tool for elucidating different intermediates while b-APP is bound to X11, especially considering the extensive sheets which form from the precursor protein and hide large surface areas of protein.
Therapeutic attempts once amyloid is formed seem unlikely to ever have striking effects. The hydrogen bonds holding the b-sheets together, in addition to the stable helix into which the sheets wind, make the amyloid so stable that dismantling it would implicate using techniques which would be physiologically damaging.
1. Dobson, Christopher. Protein misfolding,
evolution and disease. TiBS.
September 1999: 329-332.
2. Branden, Carl and John Tooze. Introduction
to Protein Structure. 2nd ed.
3. Siman, Robert and Richard W. Scott.
Strategies to alter the progression of
Alzheimer's disease. Current Opinion in Biotechnology. December 1996.
4. Lansbury, Peter T. Jr. Inhibition
of amyloid formation: a strategy to delay the onset
of Alzheimer's disease. Curr. Opinion in Chemical Biology. August 1997.
5. Shang, Shongtao et. al. Sequence-specific
recognition of the internalization motif of
the Alzheimer's amyloid precursor protein by the X11 PTB domain.
The EMBO Journal. 1997. 16: 6141-6147.
6. Muller, U. et. al. Behavioral and
anatomical deficits in mice homozygous for a
modified b-amyloid precursor protein gene. Cell. 1994. 79: 755-765.