Protein Folding and Neurodegeneration: Biophysics to the Rescue?
View Transcript of Live Discussion — Posted 26 August 2006
By David Teplow
The study of human disease usually begins at the organismal level with the identification of an abnormal phenotype. Parkinson's original description of the "Shaking Palsy" (Parkinson, 1817) or Gadjusek's report on kuru (Gajdusek and Zigas, 1957) are but two examples. Exploration of the cause(s) of a particular phenotype then leads to a deeper, cellular understanding of disease etiology. To understand and treat bacterial or viral encephalopathies, one need only identify the offending microorganism. However, for neurodegenerative diseases such as Parkinson's and the transmissible spongiform encephalopathies (TSE; the class archetype for diseases resulting from abnormal protein folding), we must work in an even deeper realm. Here, elucidation of disease etiology requires that we understand the factors controlling one of the most fundamental subcellular processes, protein folding.
Protein Folding and Neurodegeneration
Neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's, ALS, and TSE, are associated with protein folding events leading to the formation of amyloid fibrils and other pathologic protein aggregates (Prusiner, 2001). The gross histologic signs of abnormal protein folding and assembly are unmistakable-senile plaques, neurofibrillary tangles, Lewy bodies, intracellular inclusions, and spongiform degeneration. It is intuitively obvious, and well-proven experimentally, that if nascent polypeptides do not fold into their native states then they won't function. Depending on the protein and the tissues affected, abnormal folding can cause injury and death, both at the cellular and organismal levels.
Amyloid Fibril Assembly
In Alzheimer's disease, until recently, amyloid fibrils formed by the amyloid β-protein (Aβ) were considered the key pathogenetic effectors (Kirkitadze et al., 2002; Klein et al., 2001). A large body of evidence has shown that fibrils are potent neurotoxins and thus strategies to rid the body of fibrils have been pursued aggressively (Kirkitadze et al., 2002). To do so, many groups have sought to understand in greater detail how monomeric Aβ polymerizes. In 1997, two groups reported the identification and characterization of a fibril assembly intermediate, the protofibril (Harper et al., 1997; Walsh et al., 1997). This intermediate was shown to be neurotoxic (Hartley et al., 1999; Walsh et al., 1999). Subsequent studies demonstrated that many different peptides and proteins form protofibrils (Kirkitadze et al., 2002). Intermediates of this type thus appear to be a general feature of fibril assembly. In fact, the quest for deeper insight into the pathways through which fibrils form has revealed ever smaller neurotoxic assemblies (Klein, 2002). These revelations, together with in vivo studies demonstrating plaque-independent neuronal deficits in transgenic animals (Kirkitadze et al., 2002; Klein et al., 2001), have provided experimental support for a paradigm shift away from the primacy of fibrils in neurodegeneration towards the importance of oligomeric ("soluble") assemblies (Kirkitadze et al., 2002).
What Should We Study and How?
Medicinal chemists require therapeutic targets. Which of the many peptide neurotoxins described above should be targeted? This is a difficult question to answer in the absence of data establishing the relative neuropathogenic importance of each toxic assembly. However, there is one strategy that does not require this type of foreknowledge: blocking the transition from a benign, "native" protein conformation to a pathologic conformation. Doing so would prevent the sequelae of non-native folding, including the production of toxic oligomers and fibrils.
To execute this strategy, one must elucidate the intramolecular conformational states of the protein monomer and the intermolecular interactions between and among monomers. Here, it is critical to understand that intermolecular associations may moderate the intramolecular structural organization of the protein monomer. A dramatic example of this is domain swapping, a process in which analogous peptide segments from two different monomers replace each other within their "sister" peptides (Liu and Eisenberg, 2002). Domain swapping has been studied extensively in diphtheria toxin by Eisenberg et al. (Bennett et al., 1994) and also has been postulated to occur during prion amyloid formation (Knaus et al., 2001). Thus, our strategy must implement an experimental design that enables the monitoring of both intramolecular conformational dynamics and intermolecular associations.
I emphasize that the essence of the problem of pathologic protein assembly is "protein folding pathway choice." How do intrinsic structural or extrinsic environmental factors control a protein's folding pathway? Answering this question will facilitate the development of targeted pharmaceuticals, and it also promises to provide new insight into processes that are fundamental to the folding of many, and potentially all, proteins. But how do we do it? We take advantage of the broad existing armamentarium of analytical methods used successfully in basic studies of protein biophysics. General classes of these tools include nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), infrared spectroscopy (IR), circular dichroism (CD), intrinsic fluorescence, dynamic light scattering, chromatography, electrophoresis, analytical ultra-centrifugation, atomic force and electron microscopy, mass spectrometry, and molecular dynamics. These methods allow us to determine static structural (secondary, tertiary, and quaternary) features at resolutions ranging from Angstroms to micrometers.
Importantly, increasingly powerful computational approaches provide the means to convert such static structural data into a dynamic picture of the temporal changes in conformation that occur during protein folding and assembly (full text of review by Thirumalai et al., 2003). An intrinsic, and obligate, part of this latter in silico experiment is the consideration of folding thermodynamics. Proteins are dynamic structural entities that continuously sample different conformational spaces. The lifetime, i.e., the stability, of a specific conformer depends both on the free energy (?G0) of this conformational state and the transitional activation barriers to other states. These are the factors that determine protein folding pathway choice. Understanding and controlling these factors should allow us to prevent assembly of toxic structures, destabilize and eliminate existing assemblies, and protect susceptible cells from injury. Kelly et al recently provided a notable example of the potential of this approach (see related ARF story). They demonstrated that the mechanistic basis for the "anti-amyloidogenic" effect of a naturally occurring Thr119/Met amino acid substitution in transthyretin is an increase in the activation energy of the transition state between folded monomer and tetramer. They also showed that small-molecule inhibitors of fibril formation work by decreasing the free energy of the inhibitor-TTR complex, thus stabilizing this conformer.
Based on the preceding introduction, I propose the following discussion questions. The order is arbitrary and the list is incomplete. I encourage the suggestion of other important questions, as well as constructive criticism of the relevance or appropriateness of the questions themselves.
1. Transthyretin, and most other amyloidogenic proteins, has a stable native fold that has been well-determined, often crystallographically. How can we study natively unfolded proteins such as Aβ and α-synuclein? What are the questions?
2. Aβ exists not solely as a monomer, but as a mixture of small oligomers (Bitan et al., 2003; Bitan et al., 2001; Garzon-Rodriguez et al., 1997; LeVine, 1995). In addition, the oligomer distribution of Aβ differs depending on its primary structure (Bitan et al., 2003). How then can we study the structural dynamics of the monomer?
3. How can specific conformers be stabilized/destabilized?
4. How can the complexity of the in vivo milieu, with respect to its effects on Aβ, be recapitulated in vitro? Is it really important to attempt to do so?
5. How can the pathogenic importance of specific assemblies be determined?
6. What are the key questions to be addressed by in silico methods, and what assumptions should be allowed in simulations (for a recent opinion, see (Thirumalai et al., 2003))?
7. What should we target therapeutically?
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