PROTEASOME-AMYLOID · Linking aggregation of alpha-synuclein to proteasomal dysfunction; an investigation of the causes leading to Parkinson's disease

Linking aggregation of alpha-synuclein to proteasomal dysfunction; an investigation of the causes leading to Parkinson's disease

Several human diseases are linked to the formation of insoluble protein aggregates. Such assemblies are toxic to cells with the result that the tissue where aggregation occurs undergoes damage as the process proceeds. The list of such 'protein misfolding diseases' includes more than 40 human pathologies. Among these, we can find devastating neurodegenerative diseases such as Alzheimer's and Parkinson's disease (PD). In a society characterised by an ever increasing life expectancy, the social costs and impact of such pathologies is set to rise dramatically. This clearly illustrates the need for the development of new efficient therapies aimed at combatting these pathological conditions. One of the current limits to the development of innovative therapeutic tools for protein misfolding diseases is represented by the transient nature and metastability of the protein aggregates that form during the ethiopathogenesis. Thus, several intermediate species are populated along the aggregation pathway and proteins can aggregate following different mechanisms. This makes it difficult to identify the most toxic species and the mechanisms of amyloid related toxicity. Despite these difficulties, it is becoming increasingly clear that under physiological conditions a delicate equilibrium exists between protein production and degradation. Factors affecting this equilibrium may therefore lead to an excess in cellular proteins, an event which may trigger self-assembly. To get insights into this issue, in this project we decided to characterise the interplay between aggregating proteins and cellular machineries, such as the proteasome system, evolved to protect our cells from the presence of protein molecules populating misfolded and aggregation-prone conformations. In the first part of the project, we set up the required procedures to purify proteasome from fresh lysates of human cells. We subcloned genes encoding the required proteins into plasmid vectors suitable for expression in bacteria. Proteasome was purified according to well-established protocols involving affinity chromatography strategies. Next, we purified the protein whose aggregation is linked to the onset of Parkinson's disease, alpha-synuclein (aS). To reproduce the events that take place during the ethiopathogenesis of PD we induced aggregation of aS and we studied the interplay between human proteasome and aS populating a monomeric or aggregated conformation. Our results show that aggregated aS is able to interact with human proteasome and inhibit the ability of such complex to degrade standard substrates. The same activity was not shown by the monomeric protein, suggesting that the toxic effect is played only if the protein populates an amyloid conformation. In the second part of the project, we characterised the mechanism of aggregation of a model protein, the acylphosphatase fromSulfolobus solfataricus (Sso AcP). We found that Sso AcP aggregates under mildly destabilising conditions in the absence of either global or partial unfolding. When diluted into aggregation-promoting conditions, the protein experiences an increase in dynamics. This leads a specific region of the molecule to establish intermolecular interactions with an unstructured segment of another Sso AcP molecule. The latter interaction eventually triggers the aggregation process. We also set to validate in vivo the model obtained in vitro, using bacterial inclusion bodies as a model. Our results show that Sso AcP aggregates from a globally folded state in vivo as well. Furthermore, the analysis carried out on a set of protein variants with different mutations suggests that the same interaction observed in vitro is responsible for triggering aggregation in vivo. These results point towards a possible mechanism of amyloid-related toxicity based on a negative loop of proteasome activity. If toxic aggregates inhibit proteasome, this leads to a decrease in the degradation and clearance activities of such complex. The consequent increase in concentration of cellular proteins does not only represent a risk because of the possibility of misfolding events induced by wrong interactions. Our data show that aggregation can be triggered even in the absence of misfolding events. Thus, an increase in concentration may lead to the establishment of wrong interactions directly from the folded state of globular proteins and this may trigger aggregation processes. Therefore, our data may illustrate a toxic mechanism for amyloid aggregates based on the inhibition of the protein clearance and consequent protein aggregation starting from misfolded and/or folded conformational states. These results are important because they may lead to the identification of new strategies for the development of therapies against amyloid-related diseases. Furthermore, they help us in understanding how living organisms work and how cells protect themselves from the formation of deleterious aggregates.

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