PRIONS THE INFECTIOUS PROTEINS

1. PRIONS THE INFECTIOUS PROTEINS Paras Yadav*, Jaspreet Singh Arora*, Sachinandan De*, Tirtha Kumar Datta*, Surender Lal Goswami*, Aarti Bhardwaj$, Shalini Jain# and Hariom Yadav# *Animal Biotechnology, #Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India $Meerut Institute of Engeenering and Technology, Meerut, U.P., India

2. INTRODUCTION • Stanley B. Prusinercoined the term proin from Proteinaceous infective particle • and changed to prion to sound it rhythmic. • Prion diseases were caused by misfolded proteins. • Elucidated the gene and mechanism by which wild type protein bring about the • clinical disease.

3. Kuru Fatal Familial Insomnia (FFI) Creutzfeldt-Jakob disease (CJD) Scrapie Bovine Spongiform Encephalopathy (BSE) Chronic Wasting Disease (CWD) Human Animal Prion Diseases

4. Classification of prion diseases • Infectious/Exogenous • e.g., Kuru, BSE (mad cow disease), Scrapie • Spread by • Consumption of infected material. • Transfusion. • Sporadic • Familial/Hereditary • Due to autosomal dominant mutation of PrP.

5. Differences between cellular and scrapie proteins • PrPCPrPSC Solubility • Soluble Non soluble Structure • Alpha-helical Beta-sheeted Multimerisation state • Monomeric Multimeric Infectivity • Non infectious Infectious Susceptibility to Proteinase K • Susceptible Resistant

6. Steps in the biosynthesis of PrPc

7. Post-translational processing of PrP S S A C B

8. Cellular trafficking and cleavage of PrP ER Golgi Endosome

9. Mechanism of Internalization of PrPC

10. Hypothetical model for a PrPc receptor

11. Model for the function of LRP- LR as the receptor for PrP BDII (aa 53-93) Heparan sulfate chain (HS) BDI (aa 144-179) HSPG Dependent binding domain Direct binding domain (aa 161-179) LRP/LR Proteoglycan moiety Sulfated domains PrP HSPG GPI

12. Cell Culture Systems for Prion Propagation

13. Sequence of prion protein

14. Functions of Prion protein • Antioxidative • PrPC + Cu (Copper) •  • Antioxidant activity •  • Resistance to oxidative stress •  • Prevent neuronal dysfunction • (Brown et al., 2002) • Other functions

15. Models for the conversion of PrPc to PrPsc

16. Endoplasmic Reticulum Plasma membrane/ Endocytic Pathway Time taken for Transformation of mutant PrPc to a PrPSc state <10 min BFA 180C 0.5-1 hr 1-6 hr Synthesis of Mutant PrPc PIPLC- resistant Detergent insoluble Protease- resistant

17. Effect of conformation of PrP on Pro K

18. Model of the cellular pathways involved in generation of PrPSc

19. Proposed model of PrPc aggregation and induction of CtmPrP

20. Pathogenesis

21. Mechanism of PrPsc induced apoptosis

22. What are Calpains? • They generate a C-terminal fragment(C2) which has molecular weight of 27-30 kDs. • Increase in intracellular levels of Calcium increase production of terminal fragments . • Calpastatin prevents production of C2. • Inhibitors of lysosomal proteases has no effect on C2 production. Telling et al.,2004

23. Role of Caspases • It was proposed that prion-associated toxicity involves altered trafficking of PrPc. • Inhibition of ubiquitin-proteasome system(UPS). • Deposition of aggresomes of PrPSc in nerve cells. • Induction of Apoptosis with activation of Caspase 3 and Caspase 8. • Complete molecular basis for neuronal death is not known. • Aggregates of over expressed PrPc does not cause cell death. Tabrizi et al., 2005

24. Factors Responsible for Prion Propagation The AGAAAAGA Palindrome in Prion Generation Norstrom & Mastrianni, 2005

25. Factors Responsible for Prion Propagation cont… • PrPc association with lipid rafts in the early secretory pathway. • Creutzfeldt–Jakob disease (CJD) in Libyan Jews, linked to the E200K • mutation in PRNP (E200KCJD).

26. Model for chaperone-supervised PrP conversion E.g. Hsp70, GroEL

27. Factors that prevent Prion Replication • Phospholipase A2 Inhibitors prevent prion replication. • Platelet-activating Factor Antagonists also inhibits prion replication. Bate et al.,2004 • Drugs which share a N-benzylidene-benzohydrazide core structure. • Trimethylamine N-oxide (TMAO), can prevent formation of PrPSc. Bertsch et al.,2005 Bennion, 2004

28. Gross and Microscopic Changes • Gross changes • Grossly there is Cortical atrophy and ventricular dilatation may also be present.

29. Scrapie BSE Kuru CJD • Microscopic changes

30. Other microscopic changes • Gliosis within plaques. • Loss of oligodendrocytes within plaques. • Axons usually remain intact in plaques. • Both CD4+ and CD8+ lymphocytes are present in active • lesions. • (Kretzschmar et al.,1996, Wilesmith et al., 1997).

31. Diagnosis • Diagnosis can be made by: • 1. Clinical signs and Symptoms. • 2. Detection of Scrapie • Associated fibrils. • 3. Detection of Abnormal Prion protein (PrPsc) by Western blotting. • 4. Two dimensional Gel Electrophoresis. 5. Imunodiagnosis of Prion disease. • 6. Bioassay in Mice. • Scrapie Associated fibrils.

32. Mechanism of plaque formation PrPC PrPC PrPSC

33. PrPsc fibrils

34. Plaque

35. Molecular hallmark of the disorder is the accumulation of abnormal prion protein(PrPSc). • Physiological functions of cellular prion protein (PrPc) is not clear. • Identity of intracellular compartment where PrPc to PrPSc occurs is not established. • Prion peptide of 106-126 residues is found to be neurotoxic. • Studies on prion protein will open the avenues for treatment of other neurodegenerative disorders. Conclusion

36. Thank You