Towards the understanding of how chaperones function and prevent amyloidogenic diseases

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Project Summary/Abstract Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting 5.5 million Americans. Despite many decades of research there is still no known cure. AD is a protein misfolding disease, where the Alzheimer's protein, Aβ, aggregates from a random coil entity into fibrils, which are highly organized aggregates containing a cross-β sheet structure. However, the nature of the toxic species in Alzheimer's disease remains unknown. More and more attention has been given to the possibility that Aβ aggregates within mitochondria, rather than extracellular deposits of Aβ, may be responsible for the onset and progression of the disease. Nature has developed mechanisms to prevent disease-associated protein aggregation, e.g. by the introduction of heat shock proteins (Hsp's), which are overexpressed when cells undergo stress. The most important Hsp's in mitochondria are Hsp60 and Hsp70, whereas Hsp60 is the only essential chaperone in bacteria, yeast, and mammals. It is known that Hsp60 is cytoprotective against many stressors in cells and is proposed to be directly protective against AD. However nothing is known about the mechanism of how this is achieved. Since almost nothing is known about the mitochondrial Hsp60 system, including its co-chaperone Hsp10, I will use aims 1 and 2 of this proposal to explore this chaperone in absence of substrate protein. I seek to solve the structure of different states of the chaperone during its protein folding cycle by cryo-electron microscopy (cryo-EM). Further, using novel solution-state NMR methods to study sparsely-populated states, I will investigate how the co-chaperone is involved in the reaction cycle. This will provide important information on the role of Hsp10 in substrate encapsulation. Further, during my independent phase I will use the fundament knowledge obtained through aims 1 and 2 to move on to aim 3, where I will study the interaction of Hsp60 with Aβ. I will not only elucidate the structure of long-lived major states of Aβ bound to Hsp60 after substrate encapsulation by cryo-EM, but also study the transient interaction of Aβ with Hsp60 during the initial phase of the substrate folding cycle, prior to encapsulation, by solution-state NMR. These studies will provide information about which Aβ species (monomer, oligomer, or fibril) interact with Hsp60 and elucidate their structural features. Aims 1 and 2 will provide insights into fundamental questions about the mechanisms chaperones use to efficiently fold proteins into their functional forms. Further the results from aim 3 will unveil details about how Hsp60 inhibits Aβ aggregation and prevents neurodegenerative diseases, and may open up novel therapeutic strategies against Alzheimer's disease.