Chaperone-amyloid interactions

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

A prominent feature of disease pathology in many human disorders is the accumulation of amyloid aggregates of misfolded proteins. Amyloid is a highly ordered fibrous aggregate of a single type of protein that grows by recruiting the soluble form of the protein and converting it to the amyloid form as it joins an end of the fiber. Prions are an infectious class of amyloids that can be transmitted between cells and organisms. The yeast system has been used for decades to study aggregation and toxic effects of several human amyloid-forming proteins in vivo. Altering function or abundance of many different chaperones and co-chaperones or disrupting various protein quality control (PQC) processes can disrupt propagation of amyloids formed of several different proteins in a variety of ways and to varying degrees. Central among these chaperones are Hsp70s whose activities are regulated by many co-chaperones. Sis1 and Ydj1 are essential Hsp40 members of the J-domain protein family of Hsp70 co-chaperones that regulate and coordinate client substrate interactions and ATP hydrolysis. We are investigating interactions of chaperones and other PQC factors with each other and with amyloid-forming proteins to understand mechanisms of how cellular PQC systems detoxify amyloid. One aspect of our work focuses on how human chaperones, in particular J-protein regulators of Hsp70, protect cells from toxicity of polyglutamine-based amyloid toxicity and native yeast prions. We earlier showed the human J-protein DnaJB6 has potent anti-amyloid activity in yeast that, independently of Hsp70, can protect cells by disrupting propagation of yeast prions composed of different proteins. Yet, it is unable to disrupt propagation of the more toxic version of two structurally different forms of amyloid composed of one and the same protein. Thus, the ability of DnaJB6 to act directly on amyloid to halt its propagation depends more on the structure of amyloid than its amino acid composition. Further work showed that DnaJB6 could still provide modest protection from toxicity of the amyloid it could not eliminate. Our ongoing work focuses on understanding structural properties of DnaJB6 that underlie its ability to protect cells by recognizing and acting on different amyloids and different amyloid structures. DnaJB6 has an amino-terminal J-domain that interacts with Hsp70, an adjacent GF region that confers functional specificity, an ST region important for binding to amyloid, and a C-terminal region (CTD) that we earlier showed was crucial for its amyloid-detoxifying activity. Interactions among the different domains allow conformational rearrangements that determine the extent of compactness of the protein. We randomly mutagenized DnaJB6, primarily focusing on its CTD to identify locations important for DnaJB6 anti-amyloid activity. We found single amino acid alterations throughout the protein predicted to disrupt some of these interdomain interactions, and our saturation mutagenesis of the CTD identified many individual amino acid residues as critical for protecting cells from toxicity of both yeast prions and amyloids of polyglutamine. Our continued work is aimed at assessing if these substitutions have anticipated affects on the structure of the CTD and how that in turn contributes to the reduced ability of DnaJB6 to protect from amyloid toxicity. As DnaJB6 has and is being evaluated as therapeutic for polyglutamine disease, broader goals of our work are to obtain a molecular understanding of how DnaJB6 differs in its ability to recognize and antagonize amyloids of different structures. While typically propagating as a major predominant form, amyloids can contain species of less populated alternative structures. It is possible that, as with yeast prions, disrupting continued formation of the major amyloid could allow rapid accumulation of a more toxic minor species. Knowing how DnaJB6 recognizes different amyloid structures would aid in developing therapeutic strategies that could avoid such adverse consequences.