Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cell Biology
Weeks, Daniel L.
First Committee Member
Baker, Sheila A.
Second Committee Member
Dagle, John M.
Third Committee Member
Houston, Douglas W.
Fourth Committee Member
Pufall, Miles A.
Protein aggregation has long been associated with disease states, such as Alzheimer’s, Huntington’s, and Parkinson’s diseases. This model is challenged by increasing recognition of functional protein aggregates. Our focus has been exploration of intra-aggregate organization, and investigation of the ability of ATP to mediate energy independent aggregate solubilization by acting as a hydrotrope. Furthering our understanding of the composition and regulation of these structures will provide insight into how their dysregulation gives rise to disease, and provide a template for the development of novel therapeutics.
Sub-nuclear organelles are a class of non-membrane bound organelle. These compartments form through liquid-liquid phase separation of their components, resulting in a functional aggregate that demonstrates liquid properties. Many of the factors driving liquid-liquid phase separation also drive the formation of amyloid fibrils. In vitro studies have demonstrated that liquid-liquid phase separation can promote amyloid fibril formation, and that liquid-liquid phase separated droplets can mature through amyloid fibril formation. In vivo, functional amyloids are known to contribute to peptide hormone storage, melanin production, and maintenance of long-term memory. Despite these findings, there has been limited investigation into the relationship between liquid-liquid phase separated droplets and amyloid fibrils in vivo.
Our exploration of intra-aggregate organization took advantage of the numerous protein aggregates found within Xenopus oocytes. Using a dye and antibodies specific to the defining cross-β structure of amyloid, we demonstrated that amyloids form as a normal part of Xenopus oogenesis. Amyloid was detected within nuclear and cytosolic aggregates. In the cytosol, amyloid was observed within yolk platelets and a population of cortical particles whose identity has yet to be determined. In the nucleus, multiple liquid-liquid phase separated sub-nuclear organelles, including those associated with RNA polymerase I, II and III transcription as well as RNA processing, were found to have an amyloid component. Proteomic analysis of aggregate enriched material revealed proteins associated with ribonucleoprotein complex biogenesis, DNA replication, RNA processing and DNA-templated transcription. Our analysis further demonstrated that nuclear amyloids were stable, remaining intact for hours post isolation, but rapidly destabilized following treatment with RNase.
Recently, adenosine triphosphate (ATP) was demonstrated to act as a hydrotrope in vitro. Physiologic levels were shown to maintain protein solubility and solubilize recombinant liquid-liquid phase separated droplets, in the absence of ATP hydrolysis. These observations suggest that this previously unappreciated activity of ATP has a large influence on liquid-liquid phase separated droplet stability. However, these observations were made under conditions that diverge from those found in vivo. The liquid-liquid phase separated droplets tested were devoid of RNA and composed of a single protein. Amyloid containing aggregates required super physiological levels of ATP, suggesting the sub-nuclear organelles we previously described would be resistant to this activity.
By observing nucleoli within isolated Xenopus oocyte nuclei, we demonstrate that ATP has the capacity to act as a hydrotrope in vivo. However, the hydrotropic action alone of ATP is not sufficient to solubilize nucleoli. Instead, solublization requires a sensitization step which is dependent on a soluble factor(s) and requires ATP hydrolysis.
Our studies support an in vivo model of liquid-liquid phase separated aggregate solubilization where a soluble factor(s) sensitizes an aggregate in an energy dependent process. The susceptible aggregate is then solubilized by the hydrotropic action of ATP. We further speculate that liquid-liquid phase separated aggregate formation is a reversal of the above steps. These findings highlight a need to further understand the relationship between liquid-liquid phase separation and amyloid formation, and identify an urgent need to dissect the factors influencing intracellular ATP levels.
xi, 114 pages
Includes bibliographical references (pages 105-114).
Copyright © 2019 Michael Henry Hayes
Hayes, Michael Henry. "Investigating the organization and regulation of aggregated proteins within sub-nuclear organelles." PhD (Doctor of Philosophy) thesis, University of Iowa, 2019.