Associate Professor of Biological Sciences Marquette University, United States
Introduction:: According to the World Health Organization (WHO), the global cost of neurodegenerative diseases is estimated to be over $800 billion. While neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, are widespread, there are limited treatments and cures for patients. These diseases are associated with the aggregation, or clumping, of proteins in the brain, leading to neuronal cell death. However, little is known about the cellular mechanisms that contribute to the formation of these protein aggregates.
Molecular chaperones are proteins that ensure that other proteins are folded correctly. Studies have suggested that chaperones can disassemble existing aggregates, but it is unclear whether chaperones play a role in the formation of aggregates. The Hsp104 yeast molecular chaperone can disassemble existing aggregates by fragmenting them into smaller pieces. Here, we investigate whether Hsp104 has a role in aggregate formation. Based on its role as a ‘disaggregase’, we hypothesize that Hsp104 plays a role in preventing the formation of protein aggregates. Here, we have utilized both a wildtype Hsp104 and an engineered version of Hsp104, which has been shown to have higher ATP hydrolysis activity and substrate binding activity, and examine their effect on protein aggregation in yeast.
Materials and Methods:: To test our hypothesis, we used a strain of an aggregation-prone protein in yeast called Sup35 that can be monitored by colony color. Normally folded Sup35 protein yields red colonies and aggregated Sup35 yields white colonies. Yeast cells were exposed to elevated levels of wildtype Hsp104 or the engineered Hsp104 mutant and assayed for the ability to convert cells to contain aggregated Sup35 protein. Both experiments were compared to a negative empty vector control, which lacked additional Hsp104.
Results, Conclusions, and Discussions:: We found that the presence of excess wildtype Hsp104 significantly decreased the number of colonies with the aggregated Sup35 phenotype compared to the negative control (p< 0.0001). The engineered Hsp104 with higher activity also showed a significant decrease in the number of colonies with the aggregated Sup35 phenotype vs. controls (p >0.001). However, there appeared to be no difference between the wild type and engineered Hsp104.
Thus, we can conclude that Hsp104 prevents the formation of Sup35 aggregation. However, the engineered Hsp104 with higher activity did not have any advantage in preventing aggregation over the wild type.
Future studies will explore how Hsp104 prevents aggregate formation, possibly through inhibiting protein misfolding or by disassembly of small newly formed aggregates. The study of yeast chaperones may provide important therapeutic directions toward combating neurodegenerative diseases by providing a means to prevent protein aggregation.
