# Probing the Biochemical Mechanisms of Amyloid Disease

> **NIH NIH R01** · SCRIPPS RESEARCH INSTITUTE, THE · 2020 · $705,264

## Abstract

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.

## Key facts

- **NIH application ID:** 9986748
- **Project number:** 5R01DK046335-29
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** JEFFERY W KELLY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $705,264
- **Award type:** 5
- **Project period:** 1993-05-01 → 2021-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9986748

## Citation

> US National Institutes of Health, RePORTER application 9986748, Probing the Biochemical Mechanisms of Amyloid Disease (5R01DK046335-29). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9986748. Licensed CC0.

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