We seek to understand how the process of protein aggregation leads to the dysfunction and, ultimately, the death of post-mitotic tissue in the transthyretin (TTR) amyloid diseases. This understanding would enable the development of novel therapeutic strategies, the establishment of early diagnostic tactics, and the identification of biomarkers that quantify response to therapy. The TTR protein is secreted from the liver, it circulates in the blood, and its aggregation results in a primary neuropathy and/or cardiomyopathy, depending on the sequence(s) that misassembles.
In Aim 1, we aspire to understand the structure-proteotoxicity relationship(s) driving the pathology of the TTR amyloidoses. We will isolate non-native TTR structures from patient plasma and subject them to structural characterization using atomic force microscopy and negative stain electron microscopy (EM). Non-native TTR structures that decrease upon tafamidis treatment (a disease-modifying kinetic stabilizer drug that stops TTR aggregation) and exhibit relevant cellular proteotoxicity will be further structurally characterized by cryo EM and by solid-state NMR. Cytotoxicity will be assessed in relevant primary cells and C. elegans, aiming to delineate the structures that are proteotoxic and preliminary mechanistic insights, while also assessing whether there are neurotoxic vs. cardiotoxic TTR structures. Moreover, we are developing novel peptide-based probes to quantify non-native TTR structures in blood to facilitate diagnosis and response to therapy across the >100 TTR sequences linked to pathology.
In Aim 2, we will test the hypothesis that secretory pathway proteostasis network capacity in hepatocytes influences the folded structure, kinetic stability, and amyloidogenicity of secreted TTR. NMR evidence indicates the novel finding that an altered structural ensemble with enhanced TTR kinetic stability is afforded by folding TTR in a transcriptionally reprogrammed cellular proteostasis network. This aggregation resistance is due, in part, to the Hsp70 pathway, according to in vitro reconstitution experiments. We will continue to study the mechanism by which this and other proteostasis network pathways alter the structure, increase the kinetic stability, and reduce the aggregation propensity of secreted TTR in vivo. We will test the notion that wild type TTR produced by 10% of older males adopts a kinetically less stable, alternative tetramer structure that is more aggregation prone and thus leads to wild type TTR cardiomyopathy. We have developed a method to efficiently isolate TTR from healthy elderly vs. TTR amyloidosis patients to facilitate comparisons. That chaperone assisted folding can alter the folded structure of the client protein is a novel finding.
We seek to generate a structure-proteotoxicity relationship to understand how the process of protein aggregation leads to the dysfunction and, ultimately, the death of post-mitotic tissue in the transthyretin (TTR) amyloid diseases. This understanding would enable the development of novel therapeutic strategies, the establishment of early diagnostic tactics, and the identification of biomarkers that quantify response to therapy. To understand why TTR aggregates, we will test the hypothesis that the biology of the cellular protein homeostasis network strongly influences the folded structure, the kinetic stability, and amyloidogenicity of secreted TTR?thus TTR has a memory of how it was folded and this influences its aggregation propensity.
Showing the most recent 10 out of 61 publications
