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 forms 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. A class of amyloids that can be transmitted between cells and organisms (i.e., infectious) is called prions. 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 Hsp70 and Hsp104, 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 can regulate activity of the Hsp104 machinery. By incompletely attempting to resolubilize protein from prion aggregates, this machinery by default helps prions replicate in yeast. In contrast, overproducing Hsp104 results in curing cells of prions. 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 overproduced Hsp104 cures cells of prions, which has been controversial since it's discovery almost three decades ago. Some think Hsp104 cures by completely extracting monomers from prion aggregates, while others think it acts by directing retention of prion aggregates in mother cells when cells divide, thus halving the number of prion-containing cells per generation. We earlier showed that during the curing prions are evenly distributed in mother and daughter cells, which is consistent with the former mechanism. We also showed earlier that point mutations in the N-terminal region of Hsp104 disrupt its ability to cure cells of prions when overproduced, but this region is completely dispensable for its ability to propagate prions or to function in its normal cellular PQC functions. Thus, when overproduced Hsp104 depends on its N-terminal region for an activity that is specific to its ability to act on amyloid in a way that counteracts amyloid propagation in cells. We now show that a specific interaction of Hsp70 with amino acid residues in the N-terminal region of Hsp104, which overlap curing-defective mutations, is needed for prion-curing by overproduced Hsp104. Our work monitoring fluorescent prion proteins in live cells during the curing suggested the curing is mediated by dissolution of the amyloid, most likely by trimming the fibers from the ends. Our further work shows that overproduction curing by Hsp104 can occur within a single cell division, which is incompatible with the competing "asymmetric segregation" explanation. We show that many aspects of the curing, such as differential effects of Hsp70s Ssa1 and Ssa2, Hsp70 mutation that enhances curing, and dependence on the Hsp70 regulator Sti1 also hold under these conditions. All our findings are in complete agreement with a dissolution mechanism and incompatible with the asymmetric segregation of prions, leading us to conclude that when overproduced Hsp104 can interact with amyloid in a way that is determined by a specific interaction of Hsp70 and its co-chaperones that results in dissolution of the prions.