Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation

Abstract

The cellular prion protein (PrPC) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrPSc in Creutzfeldt-Jakob disease. PrPC β-endoproteolysis to the C2 fragment allows PrPSc formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, ‘S1’ and ‘S3’, locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrPSc and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrPSc and infectivity can either uncouple or engage to drive the onset of clinical disease.

Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein

“…Fig. 5A shows the lack of PrP C immunostaining in a Prnp-null homogenate compared with the spectrum obtained from homogenized TgPrP(S3.F88W)-14 brain tissue in the absence of deglycosylation; this transgenic mouse line expresses a version of S3 PrP C known as S3.F88W, and its generation has been reported previously (13). We confirmed this result using a different anti-PrP antibody known as 12B2 (19) (Fig.…”

“…Additionally, although it has been reported initially that ␤-cleavage is protease-independent, driven instead by reactive oxygen species in a copper-dependent manner (4), cleavage of recPrP by ADAM8, a member of the "a disintegrin and metalloproteinase" enzyme family, has been demonstrated in vitro (3). Recent results from our laboratory also hint at an enzymatic explanation for ␤-cleavage (13).…”

“…WT-10 and S3-3 stable cell lines were generated from the parental RK13 cell line according to methods published previously (13). WT-10 stably expresses WT murine PrP C , and S3-3 stably expresses PrP C with a mutated octapeptide repeat region that is referred to as S3 PrP C (13).…”

“…The relatively low steady-state levels of the C2 fragment can complicate analysis of ␤-cleavage events. However, a recent study from our laboratory showed that a mutant PrP C (known as "S3") with several amino acid substitutions within its octapeptide repeat domain displays accentuated ␤-cleavage in certain cell types (13). Cells expressing S3 PrP C can therefore be used to screen compounds for their effects on C1, C2, and FL PrP C levels simultaneously.…”

Prion Infectivity Plateaus and Conversion to Symptomatic Disease Originate from Falling Precursor Levels and Increased Levels of Oligomeric PrP Sc Species

“…5). For these analyses, we also included data from a low-copy number Tg.Prnp a -AL mouse line (5,22) infected with the same RML prion isolate. Since PrP C is likely a crucial factor in disease by mediating toxic signaling concentration (23), we next plotted the ratios of residual PrP C to total PrP Sc at the disease endpoint versus the corresponding incubation times using PrP Sc values from either fraction 2 (high M r ) or fraction 8 (low M r , oligomeric; Fig.…”