The Recognition, Regulation and Roles of Misfolded Proteins in the Cell
- Author(s): Smith, Melanie Hennebry
- Advisor(s): Weissman, Jonathan S
- et al.
The three dimensional structure of a protein, or its native state, determines its function. Yet, the process of folding into the native state is not straightforward; proteins can stay unfolded, misfold and even aggregate. This dissertation addresses the issue of how the cell responds to these non-native species.
In the first part, I explored the cellular regulation of a specific type of protein aggregate: prions. Prions, made from the ordered-aggregation of single proteins in non-native conformations, can exist as multiple heritable strains that have distinct structures and result in different phenotypes. Here, I investigated how the sequence of a prion protein influences its ability to propagate specific strains using the PNM2 variant of the yeast prion [PSI+]. We found that the PNM2 mutation, which prevents propagation of a specific strain, interfered with the delivery of that strain to daughter cells, illustrating that delivery is a crucial step in prion inheritance.
The remainder of this thesis addresses how the cell responds to misfolded proteins in the endoplasmic reticulum (ER). As the first part of the sectretory pathway, the ER receives all proteins destined for the extracellular space or insertion into the membrane. Some of these nascent proteins never reach their native states and are degraded by a series of pathways collectively referred to as ER-associated degradation (ERAD). In order to determine how ERAD factors accurately identify substrates for removal, I studied the substrate-binding abilities of Yos9, which in addition to its role as a lectin can also bind the protein components of substrates. I showed that Yos9 exhibits sequence specific recognition of peptides in vitro and binding of Yos9 to its substrates results in their cooperative aggregation, which is a novel phenomenon that may play a role in the regulation of ERAD.
While the response to misfolded proteins in the ER is well-studied in yeast, we sought to better understand this process in metazoans through the development of a pathway-specific screening approach that employs a reporter of the unfolded protein response. While this work is ongoing, it has the potential to reveal key mammalian-specific aspects of protein homeostasis in the ER.