PRIONS PRIONS

THE THE

INFECTIOUS PROTEINSINFECTIOUS 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

• Stanley B. Prusiner coined 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.

INTRODUCTION

• Kuru

• Fatal Familial Insomnia (FFI)

• Creutzfeldt-Jakob disease (CJD)

• Scrapie

• Bovine Spongiform Encephalopathy (BSE)

• Chronic Wasting Disease (CWD)

Prion DiseasesPrion Diseases

HumanHuman AnimalAnimal

Classification of prion diseasesClassification of prion diseases• Infectious/ExogenousInfectious/Exogenous

– e.g., Kuru, BSE (mad cow disease), Scrapie– Spread by

• Consumption of infected material.• Transfusion.

• SporadicSporadic

• Familial/HereditaryFamilial/Hereditary– Due to autosomal dominant mutation of PrP.

Differences between cellular and scrapie proteinsDifferences between cellular and scrapie proteinsPrPPrPCC PrPPrPSCSC

SolubilitySoluble Non soluble

Structure Alpha-helical Beta-sheeted

Multimerisation state Monomeric Multimeric

InfectivityNon infectious Infectious

Susceptibility to Proteinase KSusceptible Resistant

Steps in the biosynthesis of PrPSteps in the biosynthesis of PrPcc

Post-translational processing of PrPPost-translational processing of PrP

S S

AC B

ER Golgi

Endosome

Cellular trafficking and cleavage of PrPCellular trafficking and cleavage of PrP

Mechanism of Internalization of PrPMechanism of Internalization of PrPCC

Hypothetical model for a PrPHypothetical model for a PrPc c receptorreceptor

Model for the function ofModel for the function of

LRP- LR as the receptor for PrPLRP- LR as the receptor for PrP

HSPG

Dependent binding domain

Direct binding domain (aa 161-179)

LRP/LR

PrP

BDI

(aa 144-179)

Heparan sulfate chain (HS)

Sulfated domains

HSPG

Proteoglycan moiety

GPI

BDII (aa 53-93)

Cell Culture Systems for Prion PropagationCell Culture Systems for Prion Propagation

Sequence of prion proteinSequence of prion protein

PrPC + Cu (Copper)

Antioxidant activity

Resistance to oxidative stress

Prevent neuronal dysfunction

(Brown et al., 2002)

• AntioxidativeAntioxidativeFunctions of Prion proteinFunctions of Prion protein

• Other functionsOther functions

Models for the conversion of PrPc to PrPsc

Time taken for Transformation of mutant PrPTime taken for Transformation of mutant PrPc c to a PrPto a PrPScSc state state

Detergentinsoluble

PIPLC-resistant

Synthesis of Mutant PrPc

Protease-resistant

<10 min

BFA

180C

0.5-1 hr

1-6 hr

Endoplasmic Reticulum

Plasma membrane/

Endocytic Pathway

Effect of conformation of PrP on Pro KEffect of conformation of PrP on Pro K

Model of the cellular pathways Model of the cellular pathways involved in generation of PrPinvolved in generation of PrPScSc

Proposed model of PrPProposed model of PrPcc

aggregation and induction of CtmPrPaggregation and induction of CtmPrP

PathogenesisPathogenesis

Mechanism of PrPMechanism of PrPscsc induced apoptosis induced apoptosis

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

What are Calpains?What are Calpains?

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

Role of CaspasesRole of Caspases

The AGAAAAGA Palindrome in Prion Generation

Factors Responsible for Prion PropagationFactors Responsible for Prion Propagation

Norstrom & Mastrianni, 2005

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).

E.g. Hsp70, GroEL

Model for chaperone-supervised PrP conversionModel for chaperone-supervised PrP conversion

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.

Factors that prevent Prion ReplicationFactors that prevent Prion Replication

Bertsch et al.,2005

Bennion, 2004

Gross and Microscopic Changes Gross and Microscopic Changes

Grossly there is Cortical atrophy and Grossly there is Cortical atrophy and ventricular ventricular dilatation dilatation

may also be present.may also be present.

Gross changes

ScrapieScrapie BSEBSE

CJDCJDKuruKuru

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).

Other microscopic changesOther microscopic changes

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.

Diagnosis Diagnosis

Scrapie Associated fibrils.

PrPC

PrPSC

PrPC

Mechanism of plaque formationMechanism of plaque formation

PrPsc fibrils

Plaque

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.

ConclusionConclusion