Appl ID: 9981223 Grant Number : 1R21 AG067594-01 PI Name : Lander, Gabriel Grant Title : Extending the limits of cryo-EM to better understand TTR misfolding and aggregation PROJECT SUMMARY The conversion of natively folded proteins into non-functional aggregates is associated with a wide range of age-related degenerative diseases, including Alzheimer’s Disease (AD). Of great concern is the prediction that these diseases will become more prevalent in prevalence as our nation’s life expectancy increases. The development of therapeutics to prevent or reverse the protein misfolding events implicated in degenerative diseases has thus become the focus of many investigative efforts. The protein transthyretin (TTR), a thyroxine and holoretinol carrier protein exported to cerebrospinal fluid (CSF) and serum, is one such protein that demonstrates increased propensity to adopt a non-native fold and form insoluble aggregates with age. This process can occur in the native, wild type form of the TTR protein, and is responsible for wild type TTR amyloidosis (also known as senile systemic amyloidosis), which causes restrictive cardiomyopathy. Notably, there is evidence that TTR interacts with the Aβ peptide, thereby preventing Aβ fibril formation and aggregation. Studies demonstrating that the CSF of AD patients contain substantially lower concentrations of TTR than in the CSF of age-matched non-AD individuals supports a neuroprotective role of TTR. We posit that age-related TTR misfolding and aggregation abolishes the capacity of TTR to prevent Aβ fibril formation and the subsequent onset of AD. In order to better understand how a natively folded wild type TTR protein becomes predisposed to misfolding events, we seek funding to develop structural approaches to study both the native and aberrant forms of TTR. Detailed structural information regarding the destabilization and non-native oligomerization of this protein will profoundly impact our understanding of TTR misfolding and fibrillogenesis, and could lead to the development of more potent TTR stabilizing drugs that could restore neuroprotective properties of TTR in AD patients. The first aim we will push the limits of size and resolution attainable by cryo-electron microscopy to examine the high-resolution three-dimensional structures of destabilized TTR tetramers and compare them to tetramers that are stabilized by small molecule ligands, revealing how the incorporation of misfolded subunits or the improper incorporation of natively folded subunits impact TTR stability at an atomic level. The second aim will define the architecture of TTR aggregates with atomic precision in order to shed light on the assembly pathways and how different oligomeric states differentially contribute to a variety of distinct pathogeneses. Notably, since perturbations of the native folding pathway of wild type proteins are the likely cytotoxic drivers of AD, the findings of this work will likely have far-reaching impact beyond TT...