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Browsing by Author "Ross, Eric, advisor"

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    Investigating the roles prion-like domains play in cellular stress responses
    (Colorado State University. Libraries, 2018) Shattuck, Jenifer Elizabeth, author; Ross, Eric, advisor; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Telling, Glenn, committee member
    Prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates play an important role in regulating a broad-range of cellular functions. In the budding yeast Saccharomyces cerevisiae, at least 10 proteins have been identified that form self-propagating amyloid-based prions. Most known yeast prion proteins contain a low-complexity, intrinsically-disordered prion-forming domain that is converted into stable, detergent-insoluble aggregates, necessary for prion activity. These prion-forming domains tend to be glutamine/asparagine (Q/N) rich, and relatively lacking in charged and hydrophobic amino acids. To better understand the amino acid sequence features that promote prion activity, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains (PrLD). While from this study we did not identify new yeast prion proteins, we identified several PrLDs with aggregation activity. Therefore, in follow up studies we investigated the role these PrLDs play in other protein assemblies involved in cellular stress responses. First, we investigated how a prion-like protein kinase, Sky1, plays a role in regulating stress granules. Stress granules are cytoplasmic assemblies that form when translation initiation is limiting, including under a variety of stress conditions. Because these cytoplasmic granules are important regulatory machinery for cellular homeostasis, mutations that increase stress granule formation or decrease clearance have been linked to various neurodegenerative diseases. We provided evidence that Sky1 is recruited to stress granules through its aggregation-prone PrLD, and it phosphorylates an RNA-binding protein to efficiently disassemble stress granules. Additionally, we showed when Sky1 is overexpressed it can compensate for defects in other disassembly pathways. These findings contribute to understanding the regulation of stress granules, and provides a possible mechanism to mitigate persistent stress granules in neurodegenerative diseases. Next, we investigated how PrLDs are used to assemble and activate a vacuole-signaling complex. Many cellular processes are regulated primarily through the production of phosphoinositides. Specifically, synthesis and turnover of phosphatidylinositol 3,5 bisphosphate (PtdIns(3,5)P2) is regulated by a vacuole-signaling complex, containing prion-like proteins Fab1, Vac7, and Vac14. Interestingly, during hyperosmotic stress, there is a rapid and dramatic rise in PtdIns(3,5)P2, which leads to vacuole remodeling, critical for cellular survival. We used aggregation-altering mutations to characterize the role of Fab1's PrLD in response to osmotic stress. Overall, these studies provided evidence that Fab1's activation requires its aggregation prone PrLD for recruitment and efficient activation for cellular adaptation to stress. Collectively, the studies described below provide insights into the diverse roles PrLDs play in regulating cellular stress responses. Moreover, these studies have contributed to the field of aggregation-mediated cellular regulation by identifying new proteins involved, new proposed mechanisms, and new insights into the cellular consequences that arise from perturbations in regulation of these processes.
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    Investigation of the sequence features controlling aggregation or degradation of prion-like proteins
    (Colorado State University. Libraries, 2017) Cascarina, Sean Micheal, author; Ross, Eric, advisor; Ho, P. Shing, committee member; Di Pietro, Santiago, committee member; Zabel, Mark, committee member
    Protein aggregates result from the conversion of soluble proteins to an insoluble form. In some cases, protein aggregates are capable of catalyzing the conversion of their soluble protein counterparts to the insoluble form, resulting in a mode of molecular self-replication. Many of these infectious proteins, or "prions", have been identified and characterized in yeast. This has led to the development of prediction algorithms designed to identify protein domains capable of forming prions. Recently, a number human proteins with aggregation-prone prion-like domains (PrLDs) have been identified, and mutations within PrLDs have been linked to muscular and neurodegenerative disorders. However, the number and diversity of PrLD mutations linked to disease are currently limited. Therefore, the extent to which a broad assortment of PrLD mutations affect intrinsic aggregation propensity, and how well this correlates with aggregation in a cellular context, has not been systematically examined. In Chapter 2, I present evidence suggesting that our prion aggregation prediction algorithm (PAPA) is capable of predicting the effects of a diverse range of mutations on the aggregation propensity of PrLDs in vitro and in yeast. PAPA was also able to predict the effects of many but not all PrLD mutations when the protein was expressed in Drosophila, but with slightly. Therefore, while great strides have been made in predicting intrinsic aggregation propensity, a more complete understanding of the cellular factors that influence aggregation in vivo may lead to further improvement of prion prediction methods. Many intracellular protein quality control factors specialize in recognizing and degrading aggregation-prone proteins. Therefore, prions must evade or outcompete these quality control systems in order to form and propagate in a cellular context. However, the sequence features that promote degradation versus aggregation of prion domains and PrLDs have not been systematically defined. In Chapter 3, I present evidence that aggregation propensity and degradation propensity can be uncoupled in multiple ways. First, we find that only a subset of classically aggregation-promoting amino acids elicit a strong degradation response in PrLDs. Second, the amino acids that promoted degradation of the PrLDs did not induce degradation of a glutamine/asparagine (Q/N)-rich prion domain, and instead led to a dose-dependent increase in the frequency of spontaneous prion formation, suggesting that protein features surrounding aggregation-prone amino acids can modulate their ultimate effects. Furthermore, degradation suppression correlated with Q/N content of the surrounding prion domain, potentially indicating an underappreciated role for these residues in yeast prion domains. The protein features that foster susceptibility or resistance to degradation are further explored in Chapter 4. We find that Q/N-rich domains resist degradation in a primary sequence-independent manner, and can even exert a dominant degradation-inhibiting effect when coupled to a degradation-prone PrLD. Furthermore, susceptibility to degradation was a relatively de-centralized feature of the PrLD, requiring a large portion of the domain surrounding degradation-promoting amino acids to permit efficient protein turnover. Collectively, these results provide key insights into the relationship between intrinsically aggregation-prone protein features and the ability to aggregate in the context of intracellular protein quality control factors.
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    Molecular basis of [PSI+] yeast prion nucleation
    (Colorado State University. Libraries, 2013) Ben Musa, Zobaida A., author; Ross, Eric, advisor; Crans, Debbie, committee member; Zabel, Mark, committee member; Di Pietro, Santiago, committee member
    Many fatal diseases arise from the conversion of soluble, functional proteins to insoluble misfolded amyloid aggregates. Amyloid fibers are characterized by filamentous morphology, protease resistance and cross]beta structure. Prions (infectious amyloids) are a specific subset of amyloid fibers, differing from other classes of amyloids by their infectivity. Prions are found in both mammals and yeasts, but there are differences between these two groups. Most yeast prions are characterized by the presence of large numbers of glutamine and asparagine (Q/N) residues, and some other common characteristics have been noted, including the presence of few hydrophobic and charged residues. Although, several attempts have been made with limited success to develop valuable systems to predict prion activity, there is no accurate algorithm that has the ability to predict the prion-forming proteins among the Q/N-rich protein group. In the yeast, it has been shown that amino acid composition, not primary sequence, drives prion activity. Recently, preliminary efforts to define the role of amino acid composition in prion formation have been examined. The fundamental question of this project is how, in yeast Q/N-rich prions, the sequence requirements for nucleation versus propagation differ, and how this information can be used in order to develop a precise prion prediction system. By answering this question we will be able to more accurately identify additional prions in both yeast and other organisms. Our long-term goal in the comprehensive studies of prion formation and propagation mechanisms is to apply this information to mammalian prion diseases. Consequently, we will be able to identify targets for therapeutic intervention to avoid, slow-down, or reverse the development of related diseases. The study determined that the amino acids required for prion formation differ from those required for prion propagation. Identifying the sequence feature for both activities is the first step towards mechanistic studies to examine how these sequences perform their function.
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    Molecular basis of yeast prion formation
    (Colorado State University. Libraries, 2009) Toombs, James A., author; Ross, Eric, advisor
    Amyloid fibers are highly organized protein aggregates that are associated with many fatal diseases. Prions represent a unique class of amyloid fibers that are distinguished by their infectivity and inheritability. In the yeast S. cerevisiae, there are several known prion forming proteins. Since the discovery of the first yeast prions in the early 1990s, they have provided a useful model system for studying the biology of prion proteins. While it has been determined that amino acid composition is important to prion formation, there has not yet been any quantitative study aimed at determining how composition promotes or inhibits prion formation. Without this knowledge, our understanding of the events that drive prion formation and our ability to identify new prion-forming proteins is severely limited. In this dissertation, we describe our experiments with the yeast prion protein Sup35p that have illuminated the sequence requirements for yeast prion formation. From these results, we conclude that: (i) amino acid composition, not primary sequence, is the major driving force behind yeast prion propagation, and (ii) prion formation occurs in domains characterized by relatively few prion promoting residues dispersed throughout an intrinsically disordered region.
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    Understanding the role of prion-like domains in ribonucleoprotein granule dynamics
    (Colorado State University. Libraries, 2019) Boncella, Amy Elizabeth, author; Ross, Eric, advisor; Kennan, Alan, advisor; Peersen, Olve, committee member; Ackerson, Chris, committee member
    Ribonucleoprotein (RNP) granules are membraneless organelles, comprised of RNA-binding proteins and RNA, that are integrally related with the cellular stress response. Stress granules and processing bodies (p-bodies) are the two primary types of RNP granules that reversibly assemble upon stress. Interestingly, many of the proteins that localize to stress granules and p-bodies contain aggregation-prone prion-like domains (PrLDs). Furthermore, mutations in the PrLDs of a number of stress granule-associated proteins have been linked to various neurodegenerative diseases, leading to the idea that aggregation-promoting mutations in these PrLDs cause stress granule persistence. Altogether, these finding suggest an important role for these domains in the dynamics of these assemblies. In order to gain a greater understanding of how PrLDs contribute to RNP granule biology, I have taken two different approaches. The first was to investigate how aggregation-promoting mutations affect stress granule and p-body dynamics. I introduced various aggregation-promoting mutations into the PrLDs of different stress granule and p-body proteins and assessed the ability of these granules to disassemble, hypothesizing that these mutations would cause RNP granule persistence, as is observed in disease. Interestingly, despite successfully increasing the aggregation propensity of these PrLDs, stress granules and p-bodies do not persist and can efficiently disassemble after stress relief. Given that aggregation-promoting mutations in PrLDs of RNP granule proteins fail to cause granule persistence, I took a second, less targeted approach towards understanding the roles of these domains in RNP granules. I focused on investigating how PrLDs are recruited to RNP granules by screening a set of PrLDs for ability to assemble into foci upon stress. Interestingly, many PrLDs are sufficient to assemble into foci upon various stresses, with robust recruitment to stress granules upon heat shock. Furthermore, several compositional biases are observed among PrLDs that are and are not sufficient to assemble upon stress. Using these biases, we have developed a reasonably accurate composition-based predictor of PrLD recruitment into heat shock-induced stress granules, which has been further validated using rational mutation strategies. This predictor is reasonably successful at predicting whether a PrLD will assemble into stress granules upon stress. Additionally, scrambling of PrLD sequences does not disrupt recruitment to stress granules. Together, these results suggest that PrLD localization to stress granules is based on composition rather than primary sequence.
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    Yeast prion physiology
    (Colorado State University. Libraries, 2016) Nelson, Aaron C. Gonzalez, author; Ross, Eric, advisor; Woody, Robert, committee member; Peersen, Olve, committee member; Zabel, Mark, committee member
    Prions, or proteinaceous infections, are caused by proteins that have the unique ability to adopt an alternative, self-replicating structure. These self-replicating structures are the causative agent of a number of mammalian diseases including Bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, and Kuru. More recently, yeast were discovered to carry at least a dozen proteins capable of making this structural conversion. Yeast prions are unique in that their prion-forming domains are intrinsically disordered domains, with unusual compositional biases. This thesis addresses two broad questions about yeast prion physiology. First, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobics are strongly under-represented. The second chapter of this dissertation explores the effects of hydrophobic and aromatic residues on prion formation. Insertion of even a small number of hydrophobic residues is found to strongly increase prion formation. These data, combined with bioinformatics analysis of glutamine/asparagine-rich domains, suggest a limit on the number of strongly prion-promoting residues tolerated in glutamine/asparagine-rich domains. Recent studies have demonstrated that aromatic residues play a key role in the maintenance of yeast prions during cell division. Taken together, these results imply that non-aromatic hydrophobic residues are excluded from prion domains not because they inhibit prion formation, but instead because they too strongly promote aggregation, without promoting prion propagation. Despite more than 20 years of research, we still don’t know why yeast carry so many prion and prion-like domains. It has been proposed that prions may serve some biological function. Chapter Three presents progress on two lines of investigation designed to resolve this issue First, a novel bioinformatics algorithm (GARRF) is used to screen a wide range of proteomes to find examples of Q/N rich domains outside of Saccharomyces cerevisiae. Identifying other species that carry these unusual regions provides insight into their role in cellular biology. We find a wide range species carry prion-like domains at levels comparable to Saccharomyces cerevisiae, and a small number carry up to an order of magnitude more. Second, currently researchers rely primarily on yeast genetic methods to discover and monitor prions. These methods have a number of drawbacks, including a glacially slow readout time. Chapter Three reports on progress towards the development of a novel fluorescence based prion assay. This assay takes advantage of bi-molecular fluorescence complementation, a technique that uses complementary fragments of a fluorescent protein to indicate when two interacting domains are in proximity to one another. When completed, this assay will provide a means to monitor protein aggregations that is both faster and more sensitive than any existing assay.