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MISFOLDED AND AGGREGATION-PRONE PROTEINS IN NEURODEGENERATIVE DISEASES

Our approach to the problem of protein misfolding extends from in vitro approaches and single molecule studies of huntingtin and mutant SOD1 and mathematical modeling to identify different folded states and the role of molecular chaperones in bistability, in vivo studies of oligomeric states, aggregate formation, and toxicity in mammalian neuronal and non-neuronal tissue culture cells, and the development of C. elegans as a model system for the expression of aggregation-prone proteins (see C. elegans as a model system).

To address questions on the toxicity associated with protein aggregates, we are investigating how protein aggregates form in rodent and human neuronal tissue culture cell lines and in non-neuronal cells and to identify cellular proteins that associate with these misfolded and aggregate species using dynamic live cell imaging methods. Recognizing the commonality of protein misfolding, we are interested to identify the biochemical and biophysical features that are shared in addition to those characteristics that are unique. Essential cellular proteins involved in key cellular events including transcription, maintenance of cell shape and motility, protein folding, and protein degradation are associated with protein aggregates and some of these proteins are irreversibly bound whereas others are only transiently associated. Using dynamic fluorescence imaging of living cells, we have shown that huntingtin (polyglutamine) protein aggregates are dynamic structures in which glutamine-rich transcription factors, TBP or CBP, are irreversibly associated with aggregates. In contrast, the interaction between the molecular chaperones Hsp70 and Hdj1 exhibits rapid kinetics of association and dissociation similar to that observed for interactions between Hsp70 and thermally unfolded substrates. In contrast, protein aggregates formed by expression of mutant SOD1 are distinct in structure and association with cellular proteins. These studies provide new insights on the composite organization and formation of protein aggregates.

The heat shock network is an important intracellular process that protects all cells from the damage associated with protein misfolding. The principal species of the heat shock response are the molecular chaperones that mediate the process of protein quality control through interaction of the chaperones with misfolded proteins in solution or bound in aggregates. Once associated, the chaperones facilitate a protein triage decision, through interaction with various cofactors that targets its non-native substrate for refolding or to the proteasome for degradation. To understand the formation of aggregates and the protein triage process in more detail we have developed a series of steady state mathematical models of the process of aggregation and protein triage. These models include an aggregation prone protein capable of assuming three states: folded, unfolded, or aggregated. The disaggregation of the aggregated state and refolding of the unfolded state is assumed to be a function of chaperone concentration. We analyzed this simple model at steady state to understand how the parameters of the system affect the long-time behavior. As the concentration of molecular chaperones is reduced in the system it undergoes a bifurcation from one steady state to three. This bistable regime is capable of displaying both low aggregated and highly aggregated states for the same parameter values. These model-based observations of bistability may help to explain the observation of sudden transitions or discontinuities in in vitro experiments of protein aggregation, which included molecular chaperones.

References

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2. Brignull, H.R., J.F. Morley, S.M. Garcia, and R.I. Morimoto. Modeling Polyglutamine Pathogenesis in C. elegans. Methods in Enzymology 412: 256-282 (2006).
3. Finkbeiner, S., A.M. Cuervo, R.I. Morimoto and P.J. Muchowski. Disease-modifiying pathways in neurodegeneration. The Journal of Neuroscience 26(41): 10349-10357 (2006).
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