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, Aß, aggregates from a random coil entity into fibrils, which are highly organized
aggregates containing a cross-ß 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 Aß aggregates
within mitochondria, rather than extracellular deposits of Aß, 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 Aß. I will not only elucidate the structure of long-lived major states of Aß bound to
Hsp60 after substrate encapsulation by cryo-EM, but also study the transient interaction of Aß 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 Aß 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 Aß aggregation and prevents neurodegenerative
diseases, and may open up novel therapeutic strategies against Alzheimer's disease.