Live cell fluorescence resonance energy transfer predicts an altered molecular association of heterologous PrPSc with PrPC

Abstract

Prion diseases result from the accumulation of a misfolded isoform (PrP(Sc)) of the normal host prion protein (PrP(C)). PrP(Sc) propagates by templating its conformation onto resident PrP(C) to generate new PrP(Sc). Although the nature of the PrP(Sc)-PrP(C) complex is unresolved, certain segments or specific residues are thought to feature critically in its formation. The polymorphic residue 129 is one such site under considerable study. We combined transmission studies with a novel live cell yeast-based fluorescence resonance energy transfer (FRET) system that models the molecular association of PrP in a PrP(Sc)-like state, as a way to explore the role of residue 129 in this process. We show that a reduction in efficiency of prion transmission between donor PrP(Sc) and recipient PrP(C) that are mismatched at residue 129 correlates with a reduction in FRET between PrP-129M and PrP-129V in our yeast model. We further show that this effect depends on the different secondary structure propensities of Met and Val, rather than the specific amino acids. Finally, introduction of the disease-associated P101L mutation (mouse- equivalent) abolished FRET with wild-type mouse PrP, whereas mutant PrP-P101L displayed high FRET with homologous PrP-P101L, as long as residue 129 matched. These studies provide the first evidence for a physical alteration in the molecular association of PrP molecules differing in one or more residues, and they further predict that the different secondary structure propensities of Met and Val define the impaired association observed between PrP(Sc) and PrP(C) mismatched at residue 129.

FIGURE 1.

Transmission of sCJD to Tg(129V) and Tg(129M) mice. A, Western blots of rPrP^Sc from brain homogenates of the original human sCJD(129MM) and sCJD(129VV) donors and a representative Tg(129V) or Tg(129M) recipient mouse. Samples were freshly prepared from frozen frontal cortex as 10% (w/v) brain homogenate in lysis buffer. Total protein was normalized and subjected to 20 μg/ml of PK for 30 min at 37 °C, probed with monoclonal antibody 3F4. Markers on the left represent 37, 25, and 20 kDa, top to bottom. All mice were clinically sick, as defined under “Materials and Methods.” B, spongiform degeneration evident in hematoxylin and eosin (H&E) stainings of sections of the frontal cortex taken from sick Tg(129M) (left sections) and Tg(129V) (right sections) mice inoculated with brain homogenate from sCJD(129MM) (top sections) or sCJD(129VV) (bottom sections), as indicated. PrP plaques were not present in any brain region.

FIGURE 2.

PrP insolubility and PK resistance of PrP-128M and PrP-128V in the yeast cytosol. A, yeast cells expressing mouse sequence PrP-128M and PrP-128V were lysed in TEN buffer containing 10% Sarkosyl, from which equal aliquots of the total, supernatant (Sup), and pellet fractions, following 16,000 × g centrifugation, were subjected to Western analysis. PrP was probed with the C-terminal anti-mouse PrP F(ab) R1 antibody (residues 225–231). Solubility profile of each PrP was determined as the relative density of the supernatant signal relative to the total signal from each of 3 assays, using a Bio-Rad Alpha Document Imager and Quantity One® (Bio-Rad) software. The pellet represents nearly 90% of the fraction, suggesting rapid conversion of newly synthesized soluble PrP to the insoluble aggregate. B, Western blot of PrP-128M (M) and PrP-128V (V) expressed in the yeast cytosol and treated with PK (0 to 60 μg/ml) for 1 h at 37 °C. Prominent PK-resistant fragments of ∼18–20 kDa are observed at all concentrations of PK, although smaller fragments appear with higher concentrations. No major differences in conformational subtype were demonstrated by this assay.

FIGURE 3.

Colocalization of PrP-128M and PrP-128V aggregates in the yeast cytosol. Differential interference contrast (DIC) (far left) and fluorescence images of yeast after 16 h of co-expression of PrP-128M and PrP-128V fluorophore pairs tagged with either CFP or YFP, as labeled. The far right column displays merged images. Both PrP fusion proteins produce dense aggregates. Co-expression of homologous or heterologous pairs produce >90% co-aggregation.

FIGURE 4.

Live cell FRET analysis following co-expression of mouse PrP pairs homologous and heterologous at residue 128. PrP-128M::CFP was co-expressed with YFP, PrP-128M::YFP, or PrP-128V::YFP for 16 h and analyzed by FRET microscopy, as described under “Materials and Methods.” Actual Tau measures are plotted, and are recorded in Table 2. Higher Tau values represent higher FRET. Heterologous pairs displayed significantly reduced FRET compared with homologous pairs. Each bar represents the mean ± S.E. of a total of 35 to 50 separate measurements. **, p < 0.01.

FIGURE 5.

PrP-P101L associative behavior is dependent on residue 129. PrP-P101L,128M was co-expressed with PrP-P101L,128M, PrP-P101L, 128V, or WT-PrP with Met or Val at residue 128. PrP-P101L,128M was the donor (CFP tagged) and all others were acceptors (YFP tagged), as labeled. A control homologous pair of WT PrP-128M is represented by the gray bar. The CTL bar (diagonal lines) represents the τ value of the non-FRET control (PrP-P101L,128M paired with YFP), which was no different from the pairing with either WT-PrP. Each bar represents the mean ± S.E. of a total of 35 to 50 separate measurements. **, p < 0.001.

FIGURE 6.

Comparative Tau values for all PrP(128X) paired with PrP-128M and PrP-128V. A, FRET of homologous PrP(128X/X) pairs. The Tau for each homologous PrP(128X/X) pair (black bars) are represented relative to WT PrP(128M/M) (gray bar). The label below each bar indicates the amino acid substitution at residue 128. PrP(128X)::CFP donor was co-expressed with its homologous PrP(128X)::YFP acceptor, and FRET performed on 35 to 50 separate co-aggregates for each substitution. Each homologous pair resulted in reproducible and significant FRET over a non-FRET control (see Table 1). B, relative Tau of heterologous PrP(128X/V) pairs (black bars) compared with the homologous PrP(128V/V) pair (gray bar). PrP-128V::CFP was the donor and PrP(128X)::YFP the acceptor, and X is each substitution, as labeled. The non-FRET PrP(128V/YFP) is also represented (−). C, relative Tau of heterologous PrP(128X/M) pairs (black bars) relative to the homologous PrP(128M/M) pair (gray bar), with PrP-128M::CFP as donor and PrP(128X)::YFP as acceptor, and X is each substitution, as labeled. The non-FRET PrP(128M/YFP) pair is also represented (−). D, Tau values for PrP-128K paired with PrP-128K, PrP-128M, or PrP-128V, compared with the homologous PrP(128M/M) pair (black bars) and the non-FRET control (gray bars), as labeled. For all experiments in this figure, *, p < 0.05; **, p < 0.001. All values, except those in panel D, are also provided in Table 1. M, Met; A, Ala; L, Leu; I, Ile; Y, Tyr; V, Val; K, Lys.