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Amyloid Aggregation: Single Molecule Approaches to a Many Molecule Problem
Scientific summary of the Lorentz Center Workshop
Amyloid Aggregation: Single Molecule Approaches to a Many Molecule Problem, April 13 – 17 2015
Martina Huber & Vinod Subramaniam
The aggregation of proteins into amyloid nanostructures is relevant for both disease and for functional materials. Current efforts to study amyloid formation are based predominantly on bulk approaches that are not well suited to capture the dynamics and structural changes associated with individual (mis)folding transitions critical to a mechanistic understanding of the early aggregation process. This workshop focused on recent developments in experimental biophysics, including innovative single-molecule techniques, which have begun to yield unprecedented molecular and dynamic views on protein aggregation. Internationally this is an extremely timely, rapidly-developing, and relevant topic (c.f. KNAW Heineken prize 2014 to Dobson).
This workshop brought together key international players, including a mix of biophysicists, (bio)chemists, molecular modeling experts, and biologists, applying these approaches to study protein aggregation across length scales from molecules to cells and organisms. Our hope was that the workshop would
· enable an open exchange about the nuances of the latest experimental approaches, with a particular emphasis on single molecule spectroscopies, applied to protein aggregation
· establish key targets in the protein aggregation pathway that are amenable to study by these approaches
· create cross-disciplinary interactions amongst scientists from different communities and disciplines.
Invited Participants: We were exceptionally fortunate to have a number of internationally reputed scientists accept our invitation to participate in the workshop:
· Dr. Ad Bax, National Institutes of Health
· Prof. David Eliezer, Cornell University
· Prof. Thomas Jovin, Max Planck Institute for Biophysical Chemistry
· Dr. Donna Arndt-Jovin, Max Planck Institute for Biophysical Chemistry
· Dr. Reinhard Klement, Max Planck Institute for Biophysical Chemistry
· Prof. Daniel Otzen, iNano, Aarhus University
· Prof. Beat Meier, ETH Zurich
· Prof. Astrid Graslund, Stockholm University
· Dr. Philipp Selenko, Leibniz Institute for Molecular Pharmacology
· Prof. Dr. Ellen Nollen, UMC Groningen
· Prof. J. Antoinette Killian, Utrecht University
· Prof. Marc Baldus, Utrecht University
· Prof. Mariano Carrión-Vázquez, Instituto Cajal, Madrid
· Dr. Sylvestre Bonnet, Leiden University
· Prof. Teresa Head-Gordon, University of California Berkeley
· Dr. Tuomas Knowles, University of Cambridge
· Prof. Alia Matysik, University of Leipzig
· Prof. Harry Kampinga, University of Groningen
In addition, a number of senior investigators from the Dutch scientific community participated in the workshop (full list of participants available), augmented by a number of junior scientists from the Netherlands and abroad, who thoroughly enjoyed the opportunity to interact with each other.
Reflection on workshop: We held the workshop to discuss the properties of intrinsically disordered proteins and their aggregation behavior. To summarize, an analogy that came up at the last day of the workshop is particularly telling: the proteins we deal with normally resemble a mountain landscape with well-defined valleys separated by high mountain ridges. The energy landscape of intrinsically disordered proteins, on the other hand, resembles the landscape of The Netherlands. Experiment and theory is well established for the former class of landscape, the Dutch landscape-type proteins are more challenging.
The workshop atmosphere was described by all participants as unusually open with particular mention that discussions went into a depth that was well beyond what the participants would share at conferences. Interdisciplinary discussion sessions worked very well, and researchers from different backgrounds started talking to each other, which was widely appreciated by many participants. Scrutinizing biophysical results for their biomedical relevance was common and lead to new viewpoints.
At the level of the junior participants (graduate students and postdocs) the interest in their work and meeting the big-shots that they knew only from the publications was particularly valued. Most participants managed to stay throughout the workshop or at least a large portion thereof, thus providing ample opportunity to discuss questions in detail.
We found the interest in the break-out sessions remarkable and were very happy at the ample preparation that the session chairs showed.
Reflection by co-organizer Gunnar Jeschke, ETH Zürich
Now here we stand and feel derided,
the curtain falls and nothing is decided.
freely translated from Bertolt Brecht, The Good Person of Szechwan
But actually scientists feel relieved, not derided, if there still exist many open questions. The atmosphere of the Lorentz Workshop was very good. The participating scientists discussed openly and freely. Nobody was defensive about their methodology or favourite results and participants did not shy away from admitting ignorance in fields other than their own. According to my observations everybody, including the most established and renowned participants, learned a lot.
This observation on a friendly and open atmosphere does not mean that there were no controversies. We were good in agreeing to disagree, in many cases. In fact, the atmosphere was very critical. Even the most impressive results were questioned, at the very least concerning their relevance for understanding amyloid-related diseases. It was even questioned whether the topic of the workshop constitutes a (homogeneous) field of science: “An aggregate is not an aggregate is not an aggregate.” Still I think, considering how well we could talk to each other, molecular cell biologists to physical chemists, computational chemists to biochemists, biophysicists to people using genetic approaches, a common field of research certainly does exist and it became clear that such a workshop was necessary for everybody to understand the complexity of the topic.
We learned that minute details (e.g. N-acetylation of the N-terminus of -synuclein) influence structural preferences and that living cells tend to convert -synuclein to the form that they prefer, independently which form is supplied to them. We heard the hypothesis that one function (or the function?) of -synuclein might be the detoxification of peroxidated lipid molecules. We learned that, yes, fibrillization may be a defence mechanism that prolongs cell life, but that cells will die nevertheless, only a bit later. We also learned that death is overestimated, at least in the sense that even living neurons may not function anymore if they are full of amyloid aggregates, but that their function can be restored. In -synuclein structural propensity does not seem to change for residues more than two peptide bonds away from a mutation site, but for A42, introduction of a single cysteine or spin label appears to cause a drastic shift in the ensemble of conformations visited by the monomer. We don’t know yet, what is really toxic, it is probably more than one species. Cyclic D,L-peptides appear to be able to disassemble aggregates, but we don’t know how. In A, mutations that turn a certain charged residue from the inside to the outside in fibrils might be causing disease. We learned that in vitro models for membranes are rather poor mimics of biological membranes and that -synuclein happily interacts with the former, but is not seen by in-cell NMR to interact with the latter. Metal ions definitely play a role in amyloid-related disease, but are often neglected when possible mechanisms are discussed. Some conformers of amyloidogenic proteins appear to be hypermechanically stable, in fact very surprisingly stable against full stretching, but it is not known what molecular basis such stability could have. Everybody appears to agree on one thing: A1-40 and A1-42 are two very different animals. In MD simulations of intrinsically disordered proteins, the choice of force field, of water model, and the specific treatment of water-protein interaction can cause quite significant differences in structural propensities and radii of gyration. If not enough care is taken, -synuclein is more compact in silico than in experiments. Very coarse grained models reproduce some features of fibrilization, but cannot yet connected back to more detailed models. Careful kinetic experiments provide an unequivocal determination of the rate-determining step in fibril formation in vitro. Fibrils catalyse oligomer formation, which might explain that mixtures of fibrils and monomers are more toxic than either of the components alone. Secondary nucleation appears to be the culprit and can be inhibited by a certain chaperone.
I would conclude that we were treated to a lot of sophisticated experimental and theoretical approaches and to fascinating insights into detailed problems. Each piece of the puzzle is colourful, beautiful, and painted with great care in great detail. How the pieces fit together we don’t know. This may suggest that a full picture will emerge only after many additional puzzle pieces will have been found.