Calpain-dependent endoproteolytic cleavage of PrPSc modulates scrapie prion propagation.
Previous studies using post-mortem human brain extracts demonstrated that PrP in Creutzfeldt-Jakob disease (CJD) brains is cleaved by a cellular protease to generate a C-terminal fragment, referred to as C2, which has the same molecular weight as PrP-(27-30), the protease-resistant core of PrP(Sc) (1). The role of this endoproteolytic cleavage of PrP in prion pathogenesis and the identity of the cellular protease responsible for production of the C2 cleavage product has not been explored. To address these issues we have taken a combination of pharmacological and genetic approaches using persistently infected scrapie mouse brain (SMB) cells. We confirm that production of C2 is the predominant cleavage event of PrP(Sc) in the brains of scrapie-infected mice and that SMB cells faithfully recapitulate the diverse intracellular proteolytic processing events of PrP(Sc) and PrP(C) observed in vivo. While increases in intracellular calcium (Ca(2+)) levels in prion-infected cell cultures stimulate the production of the PrP(Sc) cleavage product, pharmacological inhibitors of calpains and overexpression of the endogenous calpain inhibitor, calpastatin, prevent the production of C2. In contrast, inhibitors of lysosomal proteases, caspases, and the proteasome have no effect on C2 production in SMB cells. Calpain inhibition also prevents the accumulation of PrP(Sc) in SMB and persistently infected ScN2A cells, whereas bioassay of inhibitor-treated cell cultures demonstrates that calpain inhibition results in reduced prion titers compared with control-treated cultures assessed in parallel. Our observations suggest that calpain-mediated endoproteolytic cleavage of PrP(Sc) may be an important event in prion propagation.
Yadavalli
Rajgopal
R
Department of Microbiology, University of Kentucky, Lexington, Kentucky 40536, USA.
Guttmann
Rodney P
RP
Seward
Tanya
T
Centers
Adrian P
AP
Williamson
R Anthony
RA
Telling
Glenn C
GC
D000818:Animals; D001681:Biological Assay; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000737:chemistry / Q000378:metabolism*; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D018621:PrPC Proteins / Q000378:metabolism; D018620:PrPSc Proteins / Q000378:metabolism*; D011328:Prions / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012608:Scrapie / Q000378:metabolism*; D013997:Time Factors
D002135:Calcium-Binding Proteins; D007476:Ionophores; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011328:Prions; C033668:calpastatin; D002154:Calpain; D002118:Calcium