Introduction

David Teplow led this live discussion on 30 April 2003. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
 

See also David Thirumalai's Emerging Ideas on the Molecular Basis of Protein and Peptide Aggregation" (.pdf).

Transcript:

David Teplow led a live discussion on kinetic and structural principles that govern protein (mis-/un-) folding on 30 April 2003

Participants: Dave Teplow, Harvard Medical School; Dave Holtzman, Washington University St. Louis; Keith Crutcher, University of Cincinnati; Nikolay Dokholyan, University of North Carolina Chapel Hill; Rob Tycko, NIDDK, NIH; Aleksey Lomakin, MIT; Rob Havlin, NIH; Simon Sharpe, NIH; Charlie Glabe, University of California, Irvine; Alexei Koudinov, RAMS; Liming Li, Northwestern University; Nathan Oyler, NIH; Gene Stanley, Boston University; Luis Cruz, Boston University; Brigita Urbanc, Boston University; Feng Ding, Boston University; Sergei Buldyerev, Boston University; Bogdan Tarus, Boston University; Eberhard Krause, Inst. Of Molecular Pharmacology, Berlin; David Thirumalai, University of Maryland; Saga Khare, University of North Carolina Chapel Hill; Brian Soreghan, University of Southern California; Austin Yang, University of Southern California; Alan van Giessen, Boston University.

Note: The transcript has been edited for clarity and accuracy.

Gabrielle Strobel
Perhaps we can start with a clarification. Exactly what is meant by "natively unfolded" or "intrinsically disordered"? Does it mean that a-synuclein (and also Ab) is sort of flopping about unstructured in its physiological state and then prodded into folding by a binding partner? This would fundamentally change our conventional thinking about protein structure-function relationship, no?

Dave Teplow
Gabrielle, I'll take a stab at your question and let the real biophysicists correct me. When we're taught biochemistry, we see proteins in their physiologically active forms. These forms are their native forms. The reason why Peter Lansbury and others used the term "natively unfolded" for a-synuclein and other proteins, especially peptides, is that they do not exist, to a significant degree, in the same kind of stable structure that larger native proteins like myoglobin do. This does not change our view of protein folding in the least.

Nikolay Dokholyan
I do not think there is a contradiction to the conventional thinking of protein structure-function relation. Some functions are performed by disordered protein structural parts.

Rob Tycko
Most of the liquid state NMR data, obtained on monomeric Ab, indicate that the peptide by itself is dynamically flexible in solution. In the fibril state, the peptide becomes highly ordered (at least from residue 10 on). The degree of conformational order in the prefibrillar aggregates is not yet known, I don't think. I think the issue of structural order in the prefibrillar states is an especially interesting one at this time.

Alexei Koudinov
Rob, the same may be true when the protein has its natural environment. We showed previously that Ab is bound to lipoproteins as apolipoprotein (Koudinov et al., 2001). We and others also showed that LP lipid environment affects its secondary structure (Koudinov et al., 1999).

Rob Tycko
Alexei, I'm sure you're right.

Alexei Koudinov
There is a good link to oligomers from the apolipoprotein story. Many apolipoproteins have a tendency to oligomerize and polymerize out of lipid environment, to self- and cross-associate (Koudinov et al., 1998; Koudinov, 2003). Furthermore, some apolipoproteins (OK) form different types of amyloidoses, like SAA, ApoA1, etc.

Structurally apolipoproteins depend on their amphipathicity, and Ab is also an amphipathic molecule as a recent (Apr 18) Science paper mentions and the article by Charlie Glabe of 1994 showed.(Soreghan et al., 1994).

There are several studies that show association with lipoproteins arrests toxicity of Ab. Unfortunately, last year's oligomer article by Walsh et al., (Nature 2002) and the latest Science article (April 18) miss this consideration.

Dave Teplow
Rob et al., your work in the fibril area has been superb. I agree with you that we also want to examine the earlier, less stable structures. The question is how to reduce the ensemble of structures to allow this examination.

Gabrielle Strobel
Rob, how best to study the structural order of those states?

Rob Tycko
We haven't done these measurements yet, but it should be possible to assess structural order in prefibrillar aggregates by doing solid-state NMR measurements, for example, on freeze-trapped solutions.

Dave Teplow
Yes, but what kind of data can be produced with an equilibrium mixture?

Rob Tycko
We don't know yet. If there is a very broad distribution of behavior, the data might be hard to analyze. Dave, you're the expert on preparing oligomeric Ab. What would you suggest in the way of sample preparation protocols to maximize the abundance of a single aggregated form?

Dave Teplow
We're actively working on this question. I have no "recipe" at this point, but I have two avenues of exploration: 1. Alterations in the primary structure to stabilize particular assemblies, and 2. alterations in solution conditions to achieve the same goal. I understand, but have not read, the EV work that supposedly stabilizes protofibrils. Also, Hilal Lashuel has published work on a chemical compound that stabilizes protofibrils. Maybe these attempts could work.

Gabrielle Strobel
Nikolay, do we know which functions of Ab are performed by a disordered state vs. an ordered state?

Nikolay Dokholyan
Not that I know of.

Charlie Glabe
The prefibrillar aggregates/oligomer/micelles/protofibrils/ADDLs are different from fibrils. For example, in Ab, the C-terminus is buried in oligomers and exposed in fibrils, based on fluorescence quenching.

Rob Tycko
Charlie, when you say the C-terminus is exposed in fibrils, how close to the C-terminus do you mean?

Charlie Glabe
Residue 40 in Ab 40; strange, huh? Residue 38 is also less spin-coupled and more mobile.

Gabrielle Strobel
Are there no crystal or solution structures of Ab because it is dynamically flexible? How else can we get structures of these things? Could they be bound to binding partners and then crystallized?

Nathan Oyler
Alternatively, has anyone tried dynamic light scattering on solutions of Ab as a function of time to follow the aggregation?

Charlie Glabe
A pH of 9 and lower salt concentration help stabilize oligomers; but they are not completely stable. They keep on aggregating.

Dave Teplow
Nathan, we've published extensively in this area. Aleksey Lomakin can answer all of your questions about this.

Aleksey Lomakin
I am listening.

Rob Tycko
This is why we would want to freeze-trap the oligomer samples, to prevent progressive aggregation.

Dave Teplow
Rob, what level of homogeneity would you require in the freeze-trapped SSNMR experiment?

Rob Tycko
One might be able to crystallize Ab somehow, but the conformation in the crystalline form would probably not be relevant to the conformation in oligomeric or fibrillar forms.

Charlie Glabe
Still though, oligomers are stable enough for spectroscopy. You have hours to days to work with.

Dave Teplow
Charlie, same question. How pure do the samples have to be to determine from what species the signals come?

Rob Tycko
Again, we don't really know until we try. If, for example, a given segment of Ab is helical in oligomers ranging from 10 to 50 molecules in size, we could still tell it was helical even if we had this kind of distribution.

Gabrielle Strobel
An intriguing paper earlier this month (PNAS 2003 Apr 15) suggested that the structural requirements for binding in a protein complex can overwhelm any intrinsic folding preferences within a natively unfolded protein and essentially drive its folding. Does this sound crazy?

Rob Tycko
No, it's not crazy.

Dave Holtzman
Along Gabrielle's question, are there techniques, say, by labeling Ab radioactively or fluorescently, combined with biophysical techniques, to study Ab structure in a more complex physiological solution that mimics the brain's extracellular space and its varied proteins and lipids?

Charlie Glabe
Dave, obviously, the more homogeneous the samples, the better, but you can get some idea of the structural homogeneity from the spectra.

Alexei Koudinov
Dave, this would be very important, as the solutions used seem to be too artificial to represent the in-vivo reality.

Rob Tycko
Alexei, I think one should not necessarily dismiss data obtained under "artificial" conditions without having a specific reason to do so. A lot of scientific progress has come about from in-vitro experiments.

Alexei Koudinov
You are right…. We just have to interpret them with caution and think of the next step in approaching in-vivo situation.

Gabrielle Strobel
If binding partners were involved in determining the relevant conformation, perhaps age-related changes in those other (physiological) binding proteins could have as much to do with altered Ab folding, and then aggregation, as Ab accumulation, per se. Now THIS sounds crazy, no?

Dave Teplow
Gabrielle, you're not very successful in trying to be crazy. You have suggested a very viable etiologic process.

David Thirumalai
There apparently are a number of examples where protein is natively unfolded and gets ordered only upon function, i.e., interaction with other proteins. This suggests that it is crucial to understand what unfolded means and what unfolded means in finite concentration of proteins.

Aleksey Lomakin
I always thought unfolded means no longtime correlation in structure.

Nikolay Dokholyan
Resolving a structure of a single Ab protein may provide only limited insight on "native" Ab conformation. Since it may not be stable in any of the conformations, one would get only one possible structure out of many.

Alexei Koudinov
Nikolay, it seems important to define what concentration range we are talking about. In case we make a 4>20 ng/ml PBS (or some other way buffered) will we have Ab polymerized into fibrils? Any comments? A note: 4>20 ng/ml was reported previously to be a CSF Ab concentration range.

Dave Thirumalai
I think there is considerable debate on what unfolded means in infinite dilution, let alone in the presence of other proteins/peptides.

Nikolay Dokholyan
I would define natively disordered as those peptides that do not have free energy minima of more than a couple of kBT (energy of thermal fluctuation). Of course, that does not imply that some parts of the peptide are structured….

Nikolay Dokholyan
Alexei, of course concentration affects the environment.

Dave Holtzman
Alexei, that is correct that the concentration of all Ab species in the CSF in humans ranges from ~10-30 ng/ml.

Alexei Koudinov
Dave, there are several papers on Ab concentration. Despite some variability, the trend is in the range you specify. Thanks. Gabrielle, I commented on this paper too, but it is under journal consideration. The advantage to comment at ARF is obvious in light of this delay, so I would encourage all to more actively participate in commenting on ARF news items and articles of the ARF archive.

Dave Teplow
Question for all: What do we do with Charlie's latest findings? Charlie, are you trying to define the epitope and, therefore, the "active" structure recognized by your antibody?

Charlie Glabe
Dave, yes, it would be nice to know the structure of the oligomers and what the epitope is, but it is not so easy. We (Ral Langen and I) have made some progress in [defining] the structure of the large spherical oligomers or micelles. We are trying to map the antibody-binding site by EPR spectroscopy.

Rob Tycko
Charlie, could you summarize your latest findings? I have a copy of your paper, but have not had a chance to read it yet.

Charlie Glabe
Rob, basically all soluble oligomers have a common structural feature that an antibody recognizes regardless of sequence. This is basically like Ron Wetzel's results with fibrils.

Gabrielle Strobel
All, you can read up on Charlie's paper, and comments by Dominic Walsh, Harry LeVine, and others at our ARF related news story.

Nikolay Dokholyan
I think another important outcome of Charlie's work is that immunoreactivity of Ab40 oligomer was not different from Ab42 oligomers, but delayed.

Gabrielle Strobel
Can you explain the implication of this, Nikolay?

Nikolay Dokholyan
It may be the consequence that sequence was not important for forming the oligomers, but was important for their stabilization, affecting equilibrium association.

Gabrielle Strobel
Charlie, please correct where I went wrong: I seem to recall that your synthetic Ab antigen was about 95 percent pure and you immunized with it 10 times? I find it astounding and fascinating that that produced such an interesting specificity in the polyclonal serum, a misreading of your paper?

Charlie Glabe
The peptide is about 95 percent homogeneous. The rabbit has been boosted over 20 times now and we don't see "normal" Ab antibodies being produced—same thing for mice and dogs.

Gabrielle Strobel
Wow!

Rob Tycko
I thought peptide/antibody interactions usually depend on specific side chain interactions, not just backbone conformation. Charlie, what are your possible explanations for this result that an antibody can recognize a class of structures independent of sequence?

Charlie Glabe
It must recognize the conformation of the peptide backbone. That is the only thing that they might have in common.

Alexei Koudinov
For me the Science article antibodies imply that similarity of amyloidogenesis pathways for amyloid proteins. I take it as a major take-home message (and it is in the title, as far as I remember). This importance can be of secondary value in AD, though, in case other mechanisms are of primary value (see ARF hypothesis page for other possible mechanisms). For us, such primary disease change is a cholesterol homeostasis failure (see our ARF hypothesis page).

Dave Teplow
Charlie, have you attempted to determine the kd [dissociation constant] for the antibody? This would help answer the question posed by Rob. A specific interaction would likely produce more avid binding.

Charlie Glabe
Dave, we haven't measured it accurately, but it is sufficiently high that you can do "normal" things with it like affinity purify it, IP, histochemistry.

Nikolay Dokholyan
Charlie, do you know the structure of the antibody?

Charlie Glabe
We would like to know. We tried to crystallize the Fab from rabbits, but no luck. We are trying to get a monoclonal.

Nikolay Dokholyan
What about in complex with the oligo?

Rob Tycko
If you can't solve the Fab/peptide structure by crystallography, you can get information about the bound peptide conformation from NMR. We have done this for HIV-related peptide/antibody complexes.

Charlie Glabe
Nikolay, yes, ideally, but maybe with a small peptide, too.

Nikolay Dokholyan
NMR would do, too.

Dave Thirumalai
Question for experimentalists: I don't see clean experiments that probe oligomerization kinetics as a function of protein concentration. Are there studies of this type around?

Gabrielle Strobel
The hydrophobic microenvironment of Ab, i.e., the membrane composition immediately around it, appears to influence its secondary structure. In AD age-related local alterations in the content of cholesterol, its esters, and other lipids might alter APP trafficking through the cells' various membrane compartments, and APP processing. Is there a way to model this environment in structural and aggregation studies?

Alexei Koudinov
To Gabrielle: Your note on the importance of membrane microenvironment should be tightly linked to Ab extracellular appearance as lipoprotein-associated molecule and raise a question of greatest value: What are the ways of intracellular traffic of Ab? There are some studies for this, including those discussed previously at the ARF live discussion by Gunnar Gouras.

Gabrielle Strobel
Hi Nathalie. Can you tell us about compounds Neurochem has to interrupt Ab aggregation?

Nathalie Paganini
At the moment, Neurochem is conducting a phase II clinical trial on NC-758 to investigate the safety and tolerability of this drug candidate in patients with mild to moderate Alzheimer's. NC-758 was specifically designed to modify the course of the disease by interfering with the association between glycosaminoglycans and Ab.

Gabrielle Strobel
Is this Cerebril?

Nathalie Paganini
Gabrielle, Cerebril is also targeting Ab in patients who have had lobar cerebral hemorrhage. I have to add that Cerebril is also at the stage of clinical phase II trials.

Rob Tycko
At what point in the Ab aggregation process does this glcyosaminoglycan/Ab association occur, or what is the most important point in this process, do you think?

Gabrielle Strobel
Nathalie, what is the mechanistic difference between the two compounds?

Nathalie Paganini
We are talking about the same compound.

Gabrielle Strobel
I see.

Nathalie Paganini
In the phase II trial for Cerebril, safety and tolerability are the two primary endpoints at the moment.

Dave Teplow
Following the discussion about Cerebril, it may be interesting for people to express their opinions abut which assemblies should be targeted, i.e., which assemblies are the key pathogenetic effectors in AD.

Charlie Glabe
Target any and all assemblies. If you inhibit any transition, it will be useful for structural studies.

Rob Tycko
I'm not an M.D. or even a real biologist, but isn't it established that the inflammatory response to the presence of fibrils plays at least some role in the progression of AD?

Dave Teplow
Charlie, you just lost your mulligan rights. How about sticking your neck out a little more.

Gabrielle Strobel
Yes Rob, this is how I understand it, too. And Charlie, would it not be dangerous to block some transitions when we do not know where the neurotoxicity starts? We might be accumulating toxic intermediates….

Charlie Glabe
My point is to find all inhibitors because they will all have some use. If you are talking about clinically, then you can sort out the clinically useful ones later.

Zork
How many people think that there is a single mechanism of Ab aggregation that starts with a monomer (mostly unfolded structure), and then eventually ends up as the insoluble plaques?

Dave Thirumalai
Zork, I believe that there are multiple mechanisms. We have tried to describe a couple in the COSB article in 2003.

Dave Holtzman
At least in vivo, there is evidence that Ab aggregation is not always down a single path or mechanism. When Ab aggregates in the brain of APP transgenic mice, it aggregates into fibrillar and nonfibrillar aggregates. In the same animals lacking ApoE, it aggregates almost exclusively into nonfibrillar aggregates. This doesn't rule out "one mechanism" for Ab aggregation, but suggests there may be two pathways.

Nikolay Dokholyan
Free energy landscape of multiple proteins may have several pathways from monomeric to oligomeric forms, and depending on the environment, one can imagine altering the free energy landscape so one of another pathway is mostly pronounced. So I vote for multiple pathways.

Dave Teplow
I think Nikolay has the right idea, theoretically. The question is what the relevant throughput of each pathway is, and, therefore, what its potential effect on the disease may be.

Gabrielle Strobel
Dave Thirumalai, for those who have not yet read your excellent review, can you summarize what plausible scenarios do you think govern amyloid aggregation?

Dave Thirumalai
I think we made several points. The one most relevant to scenarios is the following: The pathways depend on the nature of the monomer. If the monomer is "unfolded" natively (Ab, a-synuclein, etc.), then we argue that interaction-driven ordering is the first event prior to forming a "critical nucleus." Otherwise, one has partial denaturation followed by interaction-driven aggregation. In both instances, there are multiple pathways. But this is taken for granted nowadays.

Charlie Glabe
Even in vitro with synthetic Ab, it seems that you can elongate fibrils by addition at ends, and you can form them by "annealing" protofibrils or maybe even adding oligomers on the ends.

Aleksey Lomakin
When you inhibit some structures, Ab should go somewhere else. If we are not addressing production or sinks, we can only talk about redirection of pathways.

Nikolay Dokholyan
So the question is: How do we measure toxicity to characterize the pathways?

Alexei Koudinov
Gabrielle, it will be also important to find the differences between similar motifs in oligomers and some membrane/lipid bound proteins (including apoliproproteins in LP environment), so we would not target normal pathways.

Rob Tycko
I know it's dangerous to talk about unpublished data, but we know from recent experiments on Ab that there are at least two distinct fibril nucleation processes that lead to fibrils with distinctly different morphologies and somewhat different solid-state NMR spectra (and hence, somewhat different molecular conformations within the different morphologies). So the amino acid sequence does not uniquely determine the molecular conformation within amyloid fibrils.

Dave Teplow
Nikolay's question gets back to that of enriching particular forms of Ab.

Nikolay Dokholyan
Rob, did you observe two different pathways under the same conditions? From simulations of SH3 domain proteins, we observed that they undergo cooperative transition under one condition, while there are unpopulated intermediates under other conditions.

Dave Teplow
Rob's comment is reminiscent of the strain question in prion disease. Here, different prions can induce different folds in the same endogenous protein.

Gabrielle Strobel
All, Dave's review is available in full text atop Dave Teplow's backgrounder.

Dave Thirumalai
I think that differences in pathways might manifest in "strains" as well. This is relevant to diseases, I believe.

Gabrielle Strobel
Dave, what do you mean by strains in the context of AD?

Dave Thiurmalai
Not really in AD, but in prions.

Gabrielle Strobel
Okay.

Rob Tycko
Subtle differences in fibrillization conditions lead to different fibril morphologies, reproducibly. It seems that the key difference may be the presence or absence of interfaces (e.g., air/water interface). We plan to test for differences in fibril toxicity in cell cultures.

Dave Thirumalai
Even in AD there can be variations in the final product that depend on pathways, as Rob just mentioned.

Gabrielle Strobel
We are talking biophysics. It would be natural if many in the neurodegeneration field were facing technical and methodological barriers to engage seriously in studies of the folding of Ab, a-synuclein, tau, and other suspects. What could Alzforum do to help bring those barriers down and bring more people into this line of research? Ideas?

Dave Teplow
Rob, Dan Kirschner's work in this area has revealed that primary structure differences also result in substantial differences in morphology, for example, producing short fibrils or sheets instead of long 10 nm fibrils.

Alexei Koudinov
I'd like to call on all to check out last Friday's Science paper on nanotubes and nanowires made of Ab dipeptide (Reches and Gazit, 2002; see also comments by Claudio Soto and others). This interesting effect should be studied in greater detail.

Gabrielle Strobel
Sue Lindquist has a similar paper about prion nanotubes, and she commented briefly on the Gazit et al. paper.

Rob Tycko
In our case, the primary structure, peptide concentration, pH, temperature, ionic strength are all the same, but we still get different morphologies from different types of nucleation.

Dave Holtzman
This strain effect in prion protein diseases may be analogous to protein interactions with Ab and the risk for AD. ApoE isoform effects may be an example, via slight alterations in Ab interactions. Perhaps there are genetic alterations at other genes/proteins that modify AD risk via altering Ab conformation/pathways of folding.

Dave Thirumalai
I think an interesting question is how do different morphologies emerge and how are they related to heterogeneity of interactions (environment/sequence, etc.). This can be answered computationally as well as using toy models.

Dave Thirumalai
ApoE then may be the protein X of the prion field.

Nathalie Paganini
Interesting questions and comments that were raised. Look forward to chatting again.

Gabrielle Strobel
We have reached the end of the hour. Thank you, Dave, for your excellent backgrounder and to everyone for coming, discussing, and speculating so openly. This is fascinating and we will surely revisit the topic.

Background

Background Text
By David Teplow

Introduction
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?

References
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Gajdusek DC, Zigas V. Degenerative disease of the central nervous system in New Guinea: The endemic occurrence of kuru in the native population. New England Journal of Medicine 1957; 257: 974-978. No abstract available.

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Kirkitadze MD, Bitan G, Teplow DB. Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: The emerging role of oligomeric assemblies. Journal of Neuroscience Research 2002; 69: 567-577. Abstract

Klein WL. ADDLs & protofibrils - the missing links? Neurobiology of Aging 2002; 23: 231-233. Abstract

Klein WL, Krafft GA, Finch CE. Targeting small Aβ oligomers: the solution to an Alzheimer's disease conundrum? Trends in Neurosciences 2001; 24: 219-224. Abstract

Knaus KJ, Morillas M, Swietnicki W, Malone M, Surewicz WK, Yee VC. Crystal structure of the human prion protein reveals a mechanism for oligomerization. Nature Structural Biology 2001; 8: 770-774. Abstract

LeVine H, III. Soluble multimeric Alzheimer β(1-40) pre-amyloid complexes in dilute solution. Neurobiology of Aging 1995; 16: 755-764. Abstract

Liu Y, Eisenberg D. 3D domain swapping: As domains continue to swap. Protein Science 2002; 11: 1285-1299. Abstract

Parkinson J. An essay on the shaking palsy. London: Whittingham and Rowland, for Sherwood, Neely, and Jones, 1817.

Prusiner SB. Shattuck lecture - Neurodegenerative diseases and prions. New England Journal of Medicine 2001; 344: 1516-1526. Abstract

Thirumalai D, Klimov DK, Dima RI. Emerging ideas in the molecular basis of protein and peptide aggregation. Currrent Opinion in Structural Biology 2003; in press.

Walsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Lomakin A, et al. Amyloid β-protein fibrillogenesis - Structure and biological activity of protofibrillar intermediates. Journal of Biological Chemistry 1999; 274: 25945-25952. Abstract

Walsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB. Amyloid β-protein fibrillogenesis - Detection of a protofibrillar intermediate. Journal of Biological Chemistry 1997; 272: 22364-22372. Abstract

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References

News Citations

1. NSAIDs and Derivatives Keep Transthyretin in Its Place 31 Jan 2003
2. Amyloid Oligomer Antibody—One Size Fits All? 18 Apr 2003

Webinar Citations

1. Protein Folding and Neurodegeneration: Biophysics to the Rescue? 30 Apr 2003

Paper Citations

1. González L, Martin S, Jeffrey M. Distinct profiles of PrP(d) immunoreactivity in the brain of scrapie- and BSE-infected sheep: implications for differential cell targeting and PrP processing. J Gen Virol. 2003 May;84(Pt 5):1339-50. PubMed.
2. Bitan G, Kirkitadze MD, Lomakin A, Vollers SS, Benedek GB, Teplow DB. Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways. Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):330-5. PubMed.
3. Bitan G, Lomakin A, Teplow DB. Amyloid beta-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins. J Biol Chem. 2001 Sep 14;276(37):35176-84. PubMed.
4. Garzon-Rodriguez W, Sepulveda-Becerra M, Milton S, Glabe CG. Soluble amyloid Abeta-(1-40) exists as a stable dimer at low concentrations. J Biol Chem. 1997 Aug 22;272(34):21037-44. PubMed.
5. Hammarström P, Wiseman RL, Powers ET, Kelly JW. Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science. 2003 Jan 31;299(5607):713-6. PubMed.
6. Rao SD, Yin HZ, Weiss JH. Disruption of glial glutamate transport by reactive oxygen species produced in motor neurons. J Neurosci. 2003 Apr 1;23(7):2627-33. PubMed.
7. Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, Vassilev PM, Teplow DB, Selkoe DJ. Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J Neurosci. 1999 Oct 15;19(20):8876-84. PubMed.
8. Kirkitadze MD, Bitan G, Teplow DB. Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies. J Neurosci Res. 2002 Sep 1;69(5):567-77. PubMed.
9. Klein WL. ADDLs & protofibrils--the missing links?. Neurobiol Aging. 2002 Mar-Apr;23(2):231-5. PubMed.
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11. Eells JB. The control of dopamine neuron development, function and survival: insights from transgenic mice and the relevance to human disease. Curr Med Chem. 2003 May;10(10):857-70. PubMed.
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14. Prusiner SB. Shattuck lecture--neurodegenerative diseases and prions. N Engl J Med. 2001 May 17;344(20):1516-26. PubMed.
15. Walsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Lomakin A, Benedek GB, Selkoe DJ, Teplow DB. Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates. J Biol Chem. 1999 Sep 3;274(36):25945-52. PubMed.
16. Walsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB. Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. J Biol Chem. 1997 Aug 29;272(35):22364-72. PubMed.
17. Koudinov AR, Berezov TT, Koudinova NV. The levels of soluble amyloid beta in different high density lipoprotein subfractions distinguish Alzheimer's and normal aging cerebrospinal fluid: implication for brain cholesterol pathology?. Neurosci Lett. 2001 Nov 16;314(3):115-8. PubMed.
18. Koudinov AR, Koudinova NV, Berezov TT, Ivanov YD. HDL phospholipid: a natural inhibitor of Alzheimer's amyloid beta-fibrillogenesis?. Clin Chem Lab Med. 1999 Oct;37(10):993-4. PubMed.
19. Koudinov AR, Berezov TT, Kumar A, Koudinova NV. Alzheimer's amyloid beta interaction with normal human plasma high density lipoprotein: association with apolipoprotein and lipids. Clin Chim Acta. 1998 Feb 23;270(2):75-84. PubMed.
20. Soreghan B, Kosmoski J, Glabe C. Surfactant properties of Alzheimer's A beta peptides and the mechanism of amyloid aggregation. J Biol Chem. 1994 Nov 18;269(46):28551-4. PubMed.
21. Reches M, Gazit E. Casting metal nanowires within discrete self-assembled peptide nanotubes. Science. 2003 Apr 25;300(5619):625-7. PubMed.

Other Citations

1. David Teplow

External Citations

1. Koudinov, 2003

Further Reading