PROBING THE BIOCHEMICAL MECHANISM OF AMYLOID DISEASES

点击翻译按钮获取中文摘要

DESCRIPTION: This is the first competitive renewal following R29 funding to investigate the mechanisms of amyloid disease. The long term goals of this research program are to understand the biochemical mechanism of human amyloid disease and develop new therapeutic strategies to test hypotheses about the etiology of these diseases. The common features of human amyloid disease involve the extracellular deposition of proteins in a fiber morphology. Significant circumstantial evidence suggests that fibrils directly cause the neuropathology of the brain associated diseases and this concept has been generalized as the "amyloid hypothesis". This outstanding young investigator focuses his proposal on two amyloid diseases that do not directly involve the brain. These are familial amyloid polyneuropathy and senile systemic amyloidosis. In the familial disease, a mutated form transthyretin (prealbumin) (TTR) is deposited as fibrils in peripheral nerves or in specific organs while in the senile form of the disease normal TTR forms primarily in cardiac tissue. These diseases were chosen in order to test the general amyloid hypothesis by utilizing small molecules that can inhibit TTR amyloid fibril formation to directly assess whether inhibition of transthyretin fibril formation is sufficient to prevent the onset of amyloid disease. The first specific aim of this proposal is centered around further testing the conformational change hypothesis, which suggests that tertiary structural changes are involved to make a given protein amyloidogenic. From prior work, it was shown that TTR amyloid fibril formation results from the self-assembly of an alternative tertiary structure of the protein. This result will be extended with continued funding to apply numerous biophysical methods including mass spectrometry and NMR to precisely determine the structure of the wild type amyloidogenic intermediate, and to compare this structure to the familial amyloid polyneuropathy associated variants of transthyretin (TTR). Previous work during the past funding period has shown that these TTR variants are much less stable and denature a thousand fold faster than wild-type TTR. Under continued support, Dr. Kelly will investigate tetramers composed of mixed wild-type and familial variants to assess their stability, rates of pH mediated denaturation and amyloidogenicity. The second aim involves a structure-based drug design strategy utilizing the synergistic application of x-ray crystallography, organic synthesis and parallel screening in order to identify high affinity binding amyloid inhibitors that stabilize the native state of TTR and prevent the pH mediated conformational changes that result in fibril formation.