Investigations into Prionic Mutations Mutated Prion Protein (PrPSc) Advisor: Jamie Allison, Loveland High School Mentor: Rajiv Soman, PhD, University of Cincinnati Presenter: Jacquelyn Pohl Basics Theoretic Application Background: All of the epidemics that have affected the world’s population whether it was cancer, HPV or AIDS, have not been given a cure or prevention. Epidemics happen each day world wide. The medical facilities are ready for mild outbreaks with containment centers available, however, barely anything is known in regards to what the next big epidemic will be. There were all these pandemics and it is believed that mutated protein will be the next epidemic. Amino Acids are the building blocks for protein. Diseases Caused: Creutzfeldt-Jakob Disease Kuru Fatal Familial Insomnia Gerstmann-Sträussler-Scheinker Disease BSE – “Mad Cow Disease” Scrapie Abstract: Prions are mutated proteins and proteins are organic compounds composed of amino acids. PrPC which is the cellular version of the prion protein is located in practically all tissue in the human body however PrPSc which is the mutated version of PrPC is found solely in the brain. One theorized function of PrPC is copper mediation where as no beneficial function has been found for PrPSc. PrPSc is an infectious agent that causes many diseases based on the mutation of its amino acid sequence. It causes rapid degradation of the brain. PrPC is easily destroyed where as PrPSc is virtually indestructible. PrPC has an octarepeat region which consists of 32 amino acids. PrPC also has a cystine/cystine disulfide bond. The mutation involves the folding of the protein from one form into another. Little is known about how this mutation occurs and why it occurs. What mechanism or mechanisms are allowing this mutation to occur and how does it get started? Are there any possible solutions to end this mutation or disease? Source:http://www.phschool.com/science/biology_place/biocoach/images/translation/peptbond.gif Peptide bonds are the formation of bonds between amino acids that form the secondary structure of protein. Disease Focus: Creutzfeldt-Jakob (CJD) Rare, degenerative rapid brain disorder Three types: Sporadic: isolated occurrence Inherited: genetic Acquired: from outside body/system Symptoms: dizziness, dysarthria, myoclonus, ataxia, dementia, and death No treatment or diagnosis until autopsy Prion Protein (PrPC) Mutated Prion Protein (PrPSc) Function Theories: Copper Mediation: Copper ion regulation in the tissues of the pre-synaptic membrane of the brain. This process delivers copper (II) ions to neurons. Bone Marrow Renewal: PrPC is located on the surface of hemotopoietic stem cells as indicators that the cells will most likely become adult blood cells. Contribution to the renewal of these cells is certain however the mechanism is unknown. Involvement in Synaptic Transmission of Calcium: Loss of PrPC causes a disturbance in neuronal calcium homeostasis. It affects the calcium (II) ions release from the internal endoplasmic reticulum. This is critical because of the signaling pathways that lead to structural and functional changes in neurons. Protective Properties in Response to Oxidative Stress: PrPC helps stabilize neurons because lack of PrPC causes hypersensitivity of neurons to oxidation. O2 can act as an acid or base so copper (II) can bond with oxygen (II) and maintain homeostasis. NO BENIFICIAL FUNCTION FOUND FOR MUTATED PROTEIN PrPSc is an agent that causes three types of disorders: Infectious: From one part of the body to another Genetic: Inherited Spontaneous: Occurring with no timed execution Alpha helices are amino acid polypeptide chains in a helical shape bonded by carboxyl and amino groups. They are also bonded secondarily by hydrogen bonds causing an increased stability. Mechanism of Conversion: Cystine disulfide bond breaks due to the chemical reaction with proteinase K Sulfur free radicals interrupt the enzyme that was cutting the octarepeat allowing the region to repeat an additional 32 AA Continued rapid mutations occur because of exposed side chains and cause the symptoms of CJD Primary structure: 253 amino acids Theory for Prevention of Mutation Molecular Glue Theory: a solution that would bind to PrPSc to prevent further mutation Contain sulfur ions – to look for free radicals Contain copper (II) ions – to look for copper recycling mechanism Characteristics to avoid “rejection” from Cerebral Spinal Fluid Small quantities and concentrations because of the hypersensitivity of the brain Secondary Structure: three alpha helices and two beta sheets Conformation: 42% alpha helix and 30% beta sheet Globular domain Source:http://w3.aces.uiuc.edu/AnSci/BSE/Index_BSE_Info_at_UIUC_Science.htm Primary: 253 amino acids and the extended octarepeat region Secondary: two alpha helices and two extended beta sheets Conformation: 3% alpha helix and 43% beta sheet Beta sheets are amino acid polypeptide chains in a linear alignment bonded with hydrogen secondarily as well. Source: http://www.rothamsted.ac.uk/notebook/courses/guide/images/alpha2.gif Source:http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1QLX Cystine Disulfide bond – stabilization of alpha and beta Ionic hydrogen bonding takes place secondarily between alpha helices and beta sheets Source: http://www.learn-line.nrw.de/angebote/agenda21/bild/prion-ani-biokurs.gif PrPC to PrPSc Mutation Changes Disulfide bond breaks Octarepeat Region (8 AA) – repeats Methionine ↔ Valine (severity of the disease) Conclusion: From researching prionic mutations it has been determined that this field is very difficult, however, proteomics is very interesting and enjoyable. I look forward to continuing this work into my undergraduate and graduate work. The multiple pathways from this point just continue to increase my interest and intent to make this my life’s work. Source:http://www.brc.dcs.gla.ac.uk/~drg/courses/bioinformatics_mscIT/slides/slides5/img017.gif PrPSc Resistance Highly resistant to proteinase K – not invincible Stable through: heat, proteolysis, chemical sterilization, and autoclaving PrPC Resistance Destroyed by proteinase K Denatured by: heat, proteolysis, autoclaving, and chemical sterilization The four levels of folding for protein starts at the basic sequence and then forms alpha helices and beta sheets. Those then bind together and form a globular domain and then bind with others to form a basic protein Source: http://www.learn-line.nrw.de/angebote/agenda21/bild/prion-ani-biokurs.gif