1 / 19

Proteins as Pathogens

Stanley B. Prusiner, MD The Nobel Prize in Physiology or Medicine 1997 Presented by Shannon S. Rickner-Schmidt. Proteins as Pathogens. Definition:  Proteinaceous infectious particle, devoid of nucleic acids Affect primarily the nervous system

kieran-witt
Download Presentation

Proteins as Pathogens

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Stanley B. Prusiner, MD The Nobel Prize in Physiology or Medicine 1997 Presented by Shannon S. Rickner-Schmidt Proteins as Pathogens

  2. Definition:  • Proteinaceous infectious particle, devoid of nucleic acids • Affect primarily the nervous system • Spongiform degeneration of brain tissue • Protein accumulations in brain tissue (plaques) Prions: Causative Agents of Disease Source:Prusiner, S.B. (1998) Prions. Proceedings of the National Academy of Sciences, USA, 95; 13363-13383.

  3. Source:Pruisner, S.B. (1998) Prions. Proceedings of the National Academy of Sciences, USA, 95; 13363-13383. Sporadic (spontaneous) Infectious (transmissible) Genetic (familial) Prions: Causative Agents of Disease

  4. PrPc • Highly conserved brain glycoprotein • Normal cellular component, 35kDa • Unique biogenesis in ER Common Hydrophobic Segmentwith Distinct Epitopes, Glycosylation and Size Transmembrane Forms Secreted Form (SecPrP) Translocated into ER Lumen Prions: Causative Agents of Disease • NtmPrP • N-trans PrP • N-terminal of protein in ER lumen • CtmPrP • C-trans PrP • C-terminal of protein in ER lumen Overexpression Results in Severe Neurodegenerative Disease Source: Hegde, RS, Mastrianni, JA, Scott, MR, DeFea, KA, Tremblay, P, Torchia, M, et al (1998) A transmembrane form of the prion protein in Neurodegenerative disease. Science; 279: 827-834.

  5. PrPSc:Prion Protein Scrapie- Infectious form • When exposed to PrPSc, normal constituent of mammalian cells (CtmPrP) becomes infectious form through a structural change • Hypothesis: Ability of host to make the CtmPrP form determines effectiveness of PrPSc in causing neurodegenerative disease Function Follows Form: Isoforms

  6. Mutant mice that do not produce CtmPrP (FVB/Pmp0/0) Create transgenic (Tg) lines by introducing either mutated or normal hamster genes (SHaPrP) Correlate neurodegeneration with the PrP form expressed Designing the Experiment

  7. All Tg mice lines express PrP • WT, A117V, N108I and KH→II lines express CtmPrP • Digestion with Proteinase K results in two distinct fragments that result from CtmPrP and NtmPrP forms • ΔSTE strain is resistant to proteolysis, indicative of SecPrP WT High Levels CtmPrP Low CtmPrP No CtmPrP Measuring PrP produced by Tg mice Figure 1a: Stained with a R073, a polyclonal antibody (pAB) that recognizes all PrP.

  8. Level of PrP Expression in Brain Tissue Homogenate • Stained using monoclonal antibody (mAB) that recognizes C-terminal epitope • Confirmed different lines express different levels of CtmPrP/ SecPrP Measuring C-terminal Fragments Homogenate Amount Figure 1b: Stained with a 13A5, a monoclonal antibody that recognizes C-terminal PrP fragments.

  9. Onset of Disease Symptoms in Tg Mice Without Exposure to PrPSc • Wild Type remains asymptomatic for longer time than strains overexpressing CtmPrP Correlating CtmPrP to Disease Figure 1c: Onset of disease in un-inoculated Tg mice.

  10. PrP species found in transgenic mice • Evidence of CtmPrP in clinically ill mice • (A117V)H • (N108I)H • No evidence of CtmPrP in unaffected mice • (A117V)L • (N108I)L • No evidence of PrPSc in any mice Correlating CtmPrP to Disease Figure 1d: Stained with a R073, a polyclonal antibody that recognizes all SHa PrP.

  11. Tg Mice were Inoculated with Sc237 (SHa prions) • Correlation of Disease with CtmPrP Expression • Minimum level of CtmPrP expression necessary for disease • Within strains, increased CtmPrP expression correlates with more rapid onset of disease Susceptibility of Tg Mice to PrPSc

  12. Lines ΔSTE and (A117V)H were inoculated with PrPSc • ΔSTE: • Develops neurodegeneration much later, • Accumulates more PrPSc prior to symptom onset • Lines (KH→II)L and (KH→II)M were inoculated with PrPSc • (KH→II)L: • Develops neurodegeneration much later, • Accumulates more PrPSc prior to symptom onset Susceptibility of Tg Mice to PrPSc • Lines (A117V)L and (Av117V)H were inoculated with PrPSc • (A117V)L: • Develops neurodegeneration much later, • Accumulates more PrPSc prior to symptom onset Figure 2a-f: Propensity of Lines to Produce CtmPrP influences disease onset and PrPSc accumulation

  13. Susceptibility of Tg Mice to PrPSc Figure 2g: Ctm-index (%Ctm in Vitro x Level PrP expression) is inversely proportional to amount of PrPSc accumulated

  14. Hypothesis: PrPSc accumulation is not the proximate cause of neurodegeneration, but increased generation of CtmPrP is. • Prediction 1: • Tg mice that produce higher levels of CtmPrP do not need PrPSc to develop neurodegenerative disease, and shouldn’t be infectious • Assess transmissibility of CtmPrP-associated disease Establishing a Causal Relationship Figure 3: Prediction 1–inoculums from terminally ill mice with CtmPrP associated neurodegenerative disease or WT mice do not induce disease in null mice, Tg mice or Syrian Hamsters. Most animals live expected lifespan.

  15. Prediction 2: CtmPrP levels should rise during accumulation of PrPSc • Harder to measure directly due to assay interference Figure 4a: Solution- use Doubly Transgenic mice, expose to mouse PrPSc, which will not interfere with assay for SHa CtmPrP. • Over nine weeks, doubly Tg mice were • assayed for total PrP (pAB R073) • assayed for Syrian Hamster PrP (mAB 3F4) • Samples with no PK digestion show all mouse and SHa CtmPrP and PrPSc • Harsh PK digestion leaves PrPSc only, demonstrating no SHa PrPSc present in mice. Establishing a Causal Relationship Amount Homogenate: 1 .25 .1 .1 .25 1 Samples assayed for SHa CtmPrP, demonstrating increase over time This increase was not seen in un-inoculated mice, indicating inoculation with PrPSc caused CtmPrP increase

  16. Formation and accumulation of PrPSc • Inoculation • Spontaneous Conversion of Mutated PrPc Prion Disease: Model of Pathogenesis • CtmPrP Generation • In-trans by accumulated PrPSc • In-cis by mutations within PrP • CtmPrP mediated neurodegeneration • Misfolded protein exits ER, avoiding typical degradation process • PrPSc not necessary • CtmPrP required

  17. } Prion Disease: Future Study CtmPrP Biosynthesis & Trafficking CtmPrP Metabolism } Neurodegeneration Mechanism

  18. Horwich, A.L. & Weissman, J.S. (1997). Deadly Conformations- Protein Misfolding in Prion Disease. Cell; 89: 499-510. Prusiner, S.B. (1998). Prions. Proceedings of the National Academy of Sciences, USA; 95: 13363-13383. Prion Disease: Suggested Readings

More Related