Prions Gone Mad Insight into prion diseases: structural lessons learned from fungus Laconia SMART Team: Laura Block, Kea Schmuhl, Erin Cunningham, Tyler Foote, Ashley Toll, Cole Tidemann, Ashley Garb, Dakota Moore Advisor: Jodie Garb, Laconia High School Mentor: Anita Manogaran, Ph.D., Marquette University

Abstract Recent Developments in Prion Biology Prions in Fungi

Prion diseases, including bovine spongiform encephalopathy (mad cow) and Prion diseases are associated with protein aggregates. The aggregates form [Het-s] is a prion found in the fungus of Podospora anserina that is caused by the Het- Creutzfeldt–Jakob disease in humans, are caused by a misfolded protein in the when the normal protein undergoes misfolding (Fig. 3). This misfolded s protein. During prion formation, the Het-s protein forms ribbon-like structures brain that has the ability to convert the normal protein to the misfolded form. protein has the ability to convert other normally folded proteins of the same (Fig. 4). These ribbons then give rise to small aggregates in the cell, which are Prions in the brain lead to the formation of aggregates of misfolded protein, which kind to the misfolded form. The misfolded proteins have the tendency to associated with the [Het-s] prion. Currently, the [Het-s] prion is of interest because it are thought to be infectious and toxic to the cell. Additionally, these aggregates stack themselves in a tight arrangement called an aggregate. In humans, the is thought to have a structure similar to that of the human Prp prion. are resistant to detergents or heat, and are difficult to destroy. While reliable Prpc protein is located in the brain. The misfolding of Prpc forms large structural studies of human prion proteins have been unsuccessful, the structure aggregates within the brain and is associated with CJD. Figure 5. The [Het-s] forms ribbon- of the [HET-s] prion in the fungus Podospora anserina has been solved. The HET-s like structures during prion protein misfolds and assembles into a higher order structure called a “solenoid,” a formation5. When Het-s protein tube of circular beta sheets. Laconia SMART (Students Modeling a Research Topic) fuses to the Green Fluorescent Team modeled the HET-s protein with 3D printing technology, illustrating its Protein, long ribbon-like structures solenoid structure. Each “turn” of the solenoid consists of 21 amino acids. A are formed during the initial stages pocket of hydrophobic (V244, L276, F286, W287), hydrophilic (Q240, E280), and of [Het-s] prion formation (bottom glycine amino acid side chains is thought to stabilize the solenoid structure. Since panel). These ribbons later give human prion proteins are thought to share a similar solenoid structure with HET-s, Balguerie, A., Dos Reis, S., Coulary-Salin, B., Chaignepain, S., Sabourin, M., Schmitter, J.M., and Saupe, S.J. (2004) The sequences appended to the amyloid core region of the HET-s prion protein determine higher-order aggregate rise to fluorescent aggregates. studying HET-s provides insight into the molecular structure of human prions, Figure 3. Prion protein aggregation. A normally folded prion protein (circles) organization in vivo. J Cell Sci. 117, 2610 . which could potentially lead to advances in treatment of prion-related diseases. undergoes misfolding (becomes a triangle). The misfolded protein is able to switch a normally folded protein to the misfolded prion form (as shown by the small arrows). The accumulation of misfolded proteins leads to the formation of aggregates, which create sponge-like holes when deposited in the brain. Prions in History Structure of the [Het-s] Prion

In the past, infectious diseases have been associated with fungi, parasites, viruses, Crystal structures of the [Het-s] prion indicate that the prion has a solenoid and bacteria. In 1982, Dr. Stanley B. Prusiner suggested that prion diseases were structure, as described in Fig. 4, in which the beta sheets are “stacked”. caused by aggregating infectious proteins and won the Nobel Prize in 1997 for his Prion Aggregates Made of Beta Sheets Hydrophobic (V244, L276, F286, W287) and hydrophilic (Q240, E280) amino acids work1. Prion proteins are associated with Creuzfeldt-Jakob disease (CJD) in humans are thought to keep this spring-like structure stable. and bovine spongiform encephalopathy (BSE), also known as “mad cow” disease. The conversion of the normal protein to the misfolded form results in a Mad cow disease received wide publicity in the mid 1990’s after cattle were fed structural change. The misfolded protein is rich in beta sheets. This bone meal from prion infected sheep. The diseased cattle were then thought to secondary feature is thought to be necessary for the formation of the have entered the food supply for humans, causing an outbreak of CJD. The public aggregates. It has been proposed that the newly misfolded protein uses the outcry in response to the BSE and CJD outbreaks challenged practices on how beta sheet conformation to assemble into fibril like aggregates. nations raise and monitor meat products. In the United States, comprehensive changes resulted from a “mammalian-to-ruminant feed ban through its BSE inspection and BSE feed testing programs2.”

Figure 1. Creutzfeldt-Jakob disease and C Image modified from A B http://eebweb.arizona.edu/faculty/ma age adjusted death rate. The number of sel/research/prion- people who have died from CJD each replication/index.html Figure 6. Physical models of the [Het-s] prion based on 2KJ3.pdb. Physical protein year (blue bars) has increased between Figure 4. Beta sheets stack into a solenoid. Using the beta sheets, the models were built using ZCorp 3D printing technology. Three different angles of the 1979 and 2008, although the rate of misfolded proteins stack together to create a solenoid structure, similar to a models are shown. Hydrophobic amino acids (varying shades of blue) and death among the population remains at coil in a spring. This solenoid structure generates the fibril appearance hydrophilic amino acids (varying shades of red) involved in solenoid stabilization can approximately 1 in a million3. common in prion aggregates. be seen in A and C. The beta sheet “stacks” within the solenoid are visible in B.

"CJD (Creutzfeldt-Jakob Disease, Classic)." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 13 Dec. 2010. Web. 28 Feb. 2012.

Study of Fungal Prions Prion Diseases Lead to Sponge-like Holes in Brain Morphology Conclusion Human prion aggregates are highly resistant to heat treatment and Patients with CJD experience symptoms that include detergents. Therefore, resistance to destructive techniques and potential for Since the outbreak of bovine spongiform encephalopathy in cows and Creuzfeldt- dementia, poor motor coordination, and memory loss. infectivity make human prions possibly dangerous to work with in the Jakob disease in humans during the mid 1990’s, scientists have strived to Progression of the disease is rapid and always fatal. laboratory. Instead, prions found in organisms dissimilar to humans are used. understand prions. Because of the infectious nature of the misfolded proteins, Prions found in fungus are also caused by an infectious protein and behave researchers have looked into the fungal prion of [Het-s]. Human prions are thought Figure 2. Normal versus classic CJD brain tissue. Cross similarly to their mammalian counterparts. Using fungal prions as a model to form solenoid structures similar to [Het-s]. Therefore, studying the [Het-s] prion sections of post-mortem brains of CJD patients show large allows researchers to gain insight into the molecular structure of human structure could potentially lead scientists to new insights in relation to human holes in the tissue4. This spongy appearance is the reason prions without risking infection. Researchers have focused on the particular prions. The study of fungal prions provide a better understanding of human prions, for its descriptive name, spongiform encephalopathy. fungal prion [Het-s] of Podospora anserina . and potentially lead to the development of new medicines for prion-related

Genetic Science Learning Center. "The Mystery of diseases. Kuru." Learn.Genetics 28 February 2012

References 1. Prusiner, S.B. (1982). Science. 216, 136-144 2. Bovine Spongiform Encephalopathy, or Mad Cow Disease, Centers for Disease Control and Prevention. http://www.cdc.gov/ncidod/dvrd/bse/ 3. Creutzfeldt-Jakob Disease. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973). 4. The mystery of Kuru. Learn Genetics. Genetic Science Learning Center. University of Utah. http://learn.genetics.utah.edu/content/begin/dna/prions/kuru.html 5. Balguerie, A., et al (2004). J Cell Sci. 117, 2610 6. Van Melckebeke, H.et al (2010). J.Am.Chem.Soc. 132: 13765-13775