Title: The contribution of 14-3-3 proteins to protein aggregate homeostasis
Description: Amyloids are fibrous protein aggregates associated with age-related diseases, such as Alzheimer’s disease and Parkinson’s disease. The role of amyloids in the etiology of neurodegeneration is debatable, but genetic and molecular evidence supports a causative relationship between amyloidogenesis and disease. Amyloidogenic proteins are constitutively expressed throughout the lifespan of an organism, and yet only become pathogenic in certain situations. This led to a hunt to understand how amyloidogenic proteins could be modified in order to become aggregation-prone. One possibility that has garnered attention is phosphorylation, primarily because several amyloid aggregates such as tau and α-synuclein are often highly phosphorylated in disease. However, the contribution of phosphorylation to disease progression remains unclear.While amyloid aggregates are typically described as irreversible and pathogenic, some cells utilize reversible amyloid-like structures that serve important functions.

One example is the RNA-binding protein Rim4 which forms amyloid-like assemblies that are essential for translational control during S. cerevisiae meiosis. If Rim4 is unable to translationally repress its mRNA targets, cells mis-segregate chromosomes during meiosis resulting in aneuploid gametes. Importantly, Rim4 amyloid-like assemblies are disassembled in a phosphorylation-dependent manner at meiosis II onset which allows previously repressed transcripts to become translated. In Chapter 1, I describe the significance and complexity of protein phosphorylation as it relates to disease-associated amyloids and why Rim4 is an ideal model for studying this phenomenon. The objective of this thesis is to examine the mechanisms underlying clearance of Rim4 amyloid-like assemblies. The work described in Chapter 2 focuses on identifying co-factors that mediate clearance of amyloid-like assemblies in a physiological setting. I demonstrate that yeast 14-3-3 proteins, Bmh1 and Bmh2, bind to Rim4 assemblies and facilitate their subsequent phosphorylation and timely clearance.

Furthermore, distinct 14-3-3 proteins play non-redundant roles in facilitating phosphorylation and clearance of amyloid-like Rim4. In Chapter 3, I explore the mechanism underlying 14-3-3 contribution to Rim4 amyloid-like disassembly. I find that 14-3-3 proteins are critical for the interaction between Rim4 and its primary kinase Ime2, thus facilitating downstream multi-site phosphorylation of Rim4. In Chapter 4, I explore additional roles for 14-3-3 proteins in general protein aggregate homeostasis. I find that 14-3-3 mutants exhibit greater protein aggregate burdens. Additionally, 14-3-3 mutants accumulate ubiquitinated proteins and are sensitized to proteasome mutations, suggesting a role for 14-3-3 proteins in proteasome function. Collectively, the studies described in this thesis support a protective role for 14-3-3 proteins in protein aggregation that may have implications for amyloid biology in human disease.