Shaoman Yin

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

Mutation in the prion gene PRNP accounts for 10–15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrPC). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrPC. Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.

Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer

The cellular prion protein (PrP) is a highly conserved, widely expressed, glycosylphosphatidylinositol-anchored (GPI-anchored) cell surface glycoprotein. Since its discovery, most studies on PrP have focused on its role in neurodegenerative prion diseases, whereas its function outside the nervous system remains unclear. Here, we report that human pancreatic ductal adenocarcinoma (PDAC) cell lines expressed PrP. However, the PrP was neither glycosylated nor GPI-anchored, existing as pro-PrP and retaining its GPI anchor peptide signal sequence (GPI-PSS). We also showed that the PrP GPI-PSS has a filamin A-binding (FLNa-binding) motif and interacted with FLNa, an actin-associated protein that integrates cell mechanics and signaling. Binding of pro-PrP to FLNa disrupted cytoskeletal organization. Inhibition of PrP expression by shRNA in the PDAC cell lines altered the cytoskeleton and expression of multiple signaling proteins; it also reduced cellular proliferation and invasiveness in vitro as well as tumor growth in vivo. A subgroup of human patients with pancreatic cancer was found to have tumors that expressed pro-PrP. Most importantly, PrP expression in tumors correlated with a marked decrease in patient survival. We propose that binding of pro-PrP to FLNa perturbs FLNa function, thus contributing to the aggressiveness of PDAC. Prevention of this interaction could provide an attractive target for therapeutic intervention in human PDAC.

An Aggregation-Specific Enzyme-Linked Immunosorbent Assay: Detection of Conformational Differences between Recombinant PrP Protein Dimers and PrPSc Aggregates

ABSTRACT The conversion of the normal cellular prion protein, PrPC, into the protease-resistant, scrapie PrPSc aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrPSc aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrPSc aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimers disease or Parkinsons disease.

Prion proteins with insertion mutations have altered N-terminal conformation and increased ligand binding activity and are more susceptible to oxidative attack.

We compared the biochemical properties of a wild type recombinant normal human cellular prion protein, rPrPc, with a recombinant mutant human prion protein that has three additional octapeptide repeats, rPrP8OR. Monoclonal antibodies that are specific for the N terminus of rPrPc react much better with rPrP8OR than rPrPc, suggesting that the N terminus of rPrP8OR is more exposed and hence more available for antibody binding. The N terminus of PrPc contains a glycosaminoglycan binding motif. Accordingly, rPrP8OR also binds more glycosaminoglycan than rPrPc. In addition, the divalent cation copper modulates the conformations of rPrPc and rPrP8OR differently. When compared with rPrPc, rPrP8OR is also more susceptible to oxidative damage. Furthermore, the abnormalities associated with rPrP8OR are recapitulated, but even more profoundly, in another insertion mutant, which has five extra octapeptide repeats, rPrP10OR. Therefore, insertion mutants appear to share common features, and the degree of abnormality is proportional to the number of insertions. Any of these anomalies may contribute to the pathogenesis of inherited human prion disease.

Role of the highly conserved middle region of prion protein (PrP) in PrP-lipid interaction

Converting normal prion protein (PrP(C)) to the pathogenic PrP(Sc) isoform is central to prion disease. We previously showed that, in the presence of lipids, recombinant mouse PrP (rPrP) can be converted into the highly infectious conformation, suggesting a crucial role of lipid-rPrP interaction in PrP conversion. To understand the mechanism of lipid-rPrP interaction, we analyzed the ability of various rPrP mutants to bind anionic lipids and to gain lipid-induced proteinase K (PK) resistance. We found that the N-terminal positively charged region contributes to electrostatic rPrP-lipid binding but does not affect lipid-induced PK resistance. In contrast, the highly conserved middle region of PrP, consisting of a positively charged region and a hydrophobic domain, is essential for lipid-induced rPrP conversion. The hydrophobic domain deletion mutant significantly weakened the hydrophobic rPrP-lipid interaction and abolished the lipid-induced C-terminal PK resistance. The rPrP mutant without positive charges in the middle region reduced the amount of the lipid-induced PK-resistant rPrP form. Consistent with a critical role of the middle region in lipid-induced rPrP conversion, both disease-associated P105L and P102L mutations, localized between lysine residues in the positively charged region, significantly affected lipid-induced rPrP conversion. The hydrophobic domain-localized 129 polymorphism altered the strength of hydrophobic rPrP-lipid interaction. Collectively, our results suggest that the interaction between the middle region of PrP and lipids is essential for the formation of the PK-resistant conformation. Moreover, the influence of disease-associated PrP mutations and the 129 polymorphism on PrP-lipid interaction supports the relevance of PrP-lipid interaction to the pathogenesis of prion disease.

Test for Detection of Disease-Associated Prion Aggregate in the Blood of Infected but Asymptomatic Animals

ABSTRACT We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrPSc. However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.

A multistage pathway for human prion protein aggregation in vitro: from multimeric seeds to β-oligomers and nonfibrillar structures.

Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt-Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3-4 monomers quickly appeared. Oligomers of ~11-22 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation.

Pro-prion Binds Filamin A, Facilitating Its Interaction with Integrin β1, and Contributes to Melanomagenesis

Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin β1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin β1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin β1. Reducing PrP expression in A7 cells decreases the amount of integrin β1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin β1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.

Ligand binding promotes prion protein aggregation – role of the octapeptide repeats

Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease. PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu2+ and Zn2+. Here, we report our findings that GAG and Cu2+ promote the aggregation of recombinant human PrP (rPrP). The normal cellular prion protein has five octapeptide repeats. In the presence of either GAG or Cu2+, mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild‐type PrP. When the GAG‐binding motif, KKRPK, is deleted the effect of GAG but not that of Cu2+ is abolished. By contrast, when the Cu2+‐binding motif, the octapeptide‐repeat region, is deleted, neither GAG nor Cu2+ is able to promote aggregation. Therefore, the octapeptide‐repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand. Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti‐PrP mAbs, whereas none of the tested anti‐PrP mAbs block Cu2+‐promoted aggregation. However, a mAb that is specific for an epitope at the N‐terminus enhances aggregation in the presence of either GAG or Cu2+. Therefore, although binding of either GAG or Cu2+ promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation.

Aggregation of prion protein with insertion mutations is proportional to the number of inserts

Mutation in the prion gene, PRNP, accounts for approx. 10-15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrP(C) (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP(8OR), and the other with five more octapeptide repeats, rPrP(10OR). We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and alpha-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrP(C). Accordingly, rPrP(10OR) is also more efficient in promoting the aggregation of rPrP(C) than rPrP(8OR). These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.