Investigating the protein quality control pathways that prevent protein aggregation in the social amoeba Dictyostelium discoideum

Abstract

A major hallmark of many neurodegenerative diseases is protein aggregation. Protein aggregation occurs in part due to failure of protein quality control pathways. A better understanding of these pathways could allow us to improve therapies to help control protein aggregation. Here, I investigated molecular chaperones of the social amoeba, Dictyostelium discoideum, an organism resistant to polyglutamine (polyQ) protein aggregation and whose proteostasis pathways have not been intensively studied. Serine- rich chaperone protein 1 (SRCP1), a Dictyostelium-specific chaperone, was previously found to prevent aggregation of exon 1 of the mutant huntingtin protein (Htt) in both Dictyostelium and human cells. I discovered that a region within its C-terminus, residues 61-80 (SRCP161-80), were sufficient to suppress Htt aggregation in vitro and in human cells. While I was unable to purify recombinant protein for structural studies, we utilized computational and experimental approaches to show that SRCP1 is predicted to be a mainly disordered protein with residues 61-80 structured in a β-hairpin conformation. Additionally, I found that a peptide of residues 61-70 were the minimal sequence required for SRCP1 to suppress aggregation, and a tandem repeat peptide of this sequence increased its potency. Kinetic analysis revealed that SRCP161-80 inhibits aggregation of mutant Htt at the secondary nucleation step, and I demonstrated that SRCP161-80 does not inhibit pure polyglutamine (polyQ) aggregation, suggesting it interacts with another part of the Htt protein. Finally, I show that SRCP161-80 is also sufficient to suppress aggregation of another polyQ protein, mutant ataxin-3, confirming that SRCP161-80 can generally inhibit aggregation. Together, these data give a clearer image of both SRCP1’s structure and function and could help develop therapies for neurodegenerative diseases. Another Dictyostelium protein proposed to prevent polyQ aggregation is heat shock protein 101 (Hsp101). Hsp101 was previously shown to act as a protein disaggregase of heat-induced polyQ foci. To further investigate Hsp101 as a polyQ disaggregase, I generated Hsp101 knockout Dictyostelium strains and found that Hsp101 knockout did not cause the accumulation of mutant Htt or prion aggregates under heat stress, suggesting that Hsp101 does not regulate protein aggregation. I instead turned to determine the biological role of Hsp101 in Dictyostelium. I showed that the only Hsp101 knockout phenotype was an axenic growth defect, which could be rescued by Hsp101 overexpression. I also used mass spectrometry of Hsp101 pulldowns to reveal high fold enrichments for proteins involved in the Skp1-Cullin-F-box (SCF) ubiquitin ligase pathway. These results suggest that Hsp101 may play a role in the SCF pathway, and could uncover novel roles for Hsp100-type chaperones.