Persistent ER stress causes GPI anchor deficit to convert a GPI-anchored prion protein into pro-PrP via the ATF6-miR449c-5p-PIGV axis.

Endoplasmic reticulum (ER) stress and unfolded protein response are cells' survival strategies to thwart disruption of proteostasis. Tumor cells are continuously being challenged by ER stress. The prion protein, PrP, normally a glycosylphosphatidylinositol (GPI)-anchored protein exists as a pro-PrP retaining its GPI-peptide signal sequence in human pancreatic ductal cell adenocarcinoma (PDAC). Higher abundance of pro-PrP indicates poorer prognosis in PDAC patients. The reason why PDAC cells express pro-PrP is unknown. Here, we report that persistent ER stress causes conversion of GPI-anchored PrP to pro-PrP via a conserved ATF6-miRNA449c-5p-PIGV axis. Mouse neurons and AsPC-1, a PDAC cell line, express GPI-anchored PrP. However, continuous culture of these cells with the ER stress inducers thapsigargin or brefeldin A results in the conversion of a GPI-anchored PrP to pro-PrP. Such a conversion is reversible; removal of the inducers allows the cells to re-express a GPI-anchored PrP. Mechanistically, persistent ER stress increases the abundance of an active ATF6, which increases the level of miRNA449c-5p (miR449c-5p). By binding the mRNA of PIGV at its 3'-UTRs, miR449c-5p suppresses the level of PIGV, a mannosyltransferase pivotal in the synthesis of the GPI anchor. Reduction of PIGV leads to disruption of the GPI anchor assembly, causing pro-PrP accumulation and enhancing cancer cell migration and invasion. The importance of ATF6-miR449c-5p-PIGV axis is recapitulated in PDAC biopsies as the higher levels of ATF6 and miR449c-5p and lower levels of PIGV are markers of poorer outcome for patients with PDAC. Drugs targeting this axis may prevent PDAC progression.

Pro-prion, as a membrane adaptor protein for E3 ligase c-Cbl, facilitates the ubiquitination of IGF-1R, promoting melanoma metastasis.

Aberrant activation of receptor tyrosine kinase (RTK) is usually a result of mutation and plays important roles in tumorigenesis. How RTK without mutation affects tumorigenesis remains incompletely understood. Here we show that in human melanomas pro-prion (pro-PrP) is an adaptor protein for an E3 ligase c-Cbl, enabling it to polyubiquitinate activated insulin-like growth factor-1 receptor (IGF-1R), leading to enhanced melanoma metastasis. All human melanoma cell lines studied here express pro-PrP, retaining its glycosylphosphatidylinositol-peptide signal sequence (GPI-PSS). The sequence, PVILLISFLI in the GPI-PSS of pro-PrP, binds c-Cbl, docking c-Cbl to the inner cell membrane, forming a pro-PrP/c-Cbl/IGF-1R trimeric complex. Subsequently, IGF-1R polyubiquitination and degradation are augmented, which increases autophagy and tumor metastasis. Importantly, the synthetic peptide PVILLISFLI disrupts the pro-PrP/c-Cbl/IGF-1R complex, reducing cancer cell autophagy and mitigating tumor aggressiveness in vitro and in vivo. Targeting cancer-associated GPI-PSS may provide a therapeutic approach for treating human cancers expressing pro-PrP.

GPI-anchored ligand-BioID2-tagging system identifies Galectin-1 mediating Zika virus entry.

Identification of host factors facilitating pathogen entry is critical for preventing infectious diseases. Here, we report a tagging system consisting of a viral receptor-binding protein (RBP) linked to BioID2, which is expressed on the cell surface via a GPI anchor. Using VSV or Zika virus (ZIKV) RBP, the system (BioID2- RBP(V)-GPI; BioID2-RBP(Z)-GPI) faithfully identifies LDLR and AXL, the receptors of VSV and ZIKV, respectively. Being GPI-anchored is essential for the probe to function properly. Furthermore, BioID2-RBP(Z)-GPI expressed in human neuronal progenitor cells identifies galectin-1 on cell surface pivotal for ZIKV entry. This conclusion is further supported by antibody blocking and galectin-1 silencing in A549 and mouse neural cells. Importantly, Lgals1 -/- mice are significantly more resistant to ZIKV infection than Lgals1 +/+ littermates are, having significantly lower virus titers and fewer pathologies in various organs. This tagging system may have broad applications for identifying protein-protein interactions on the cell surface.

Targeting type I collagen for cancer treatment.

Collagen is the most abundant protein in animals. Interactions between tumor cells and collagen influence every step of tumor development. Type I collagen is the main fibrillar collagen in the extracellular matrix and is frequently upregulated during tumorigenesis. The binding of type I collagen to its receptors on tumor cells promotes tumor cell proliferation, epithelial-mesenchymal transition and metastasis. Type I collagen also regulates the efficacy of tumor therapies, such as chemotherapy, radiotherapy and immunotherapy. Furthermore, type I collagen fragments are diagnostic markers of metastatic tumors and have prognostic value. Inhibition of type I collagen synthesis has been reported to have antitumor effects in animal models. However, collagen has also been shown to possess antitumor activity. Therefore, the roles that type I collagen plays in tumor biology are complex and tumor type-dependent. In this review, we discuss the expression and regulation of synthesis of type I collagen, as well as the role upregulated type I collagen plays in various stages of cancer progression. We also discuss the role of collagen in tumor therapy. Finally, we highlight several recent approaches targeting type I collagen for cancer treatment.

Crizotinib and Doxorubicin Cooperatively Reduces Drug Resistance by Mitigating MDR1 to Increase Hepatocellular Carcinoma Cells Death.

As the sixth most lethal cancers worldwide, hepatocellular carcinoma (HCC) has been treated with doxorubicin (Dox) for decades. However, chemotherapy resistance, especially for Dox is an even more prominent problem due to its high cardiotoxicity. To find a regimen to reduce Dox resistance, and identify the mechanisms behind it, we tried to identify combination of drugs that can overcome drug resistance by screening tyrosine kinase inhibitor(s) with Dox with various HCC cell lines in vitro and in vivo. We report here that combination of Crizo and Dox has a synergistic effect on inducing HCC cell death. Accordingly, Crizo plus Dox increases Dox accumulation in nucleus 3-16 times compared to Dox only; HCC cell death enhanced at least 50% in vitro and tumor weights reduced ranging from 35 to 65%. Combining these two drugs reduces multiple drug resistance 1 (MDR1) protein as a result of activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), which phosphorylates eIF2α, leading to protein translational repression. Additionally, PERK stimulation activates C-Jun terminal kinase (JNK), resulting in accumulation of unfused autophagosome to enhance autophagic cell death via Poly-ADP-ribosyltransferase (PARP-1) cleavage. When the activity of PERK or JNK is blocked, unfused autophagosome is diminished, cleaved PARP-1 is reduced, and cell death is abated. Therefore, Crizo plus Dox sensitize HCC drug resistance by engaging PERK-p- eIF2α-MDR1, and kill HCC cells by engaging PERK-JNK- autophagic cell death pathways. These newly discovered mechanisms of Crizo plus Dox not only provide a potential treatment for HCC but also point to an approach to overcome MDR1 related drug resistance in other cancers.

Tumor Necrosis Factor α Reduces SNAP29 Dependent Autolysosome Formation to Increase Prion Protein Level and Promote Tumor Cell Migration.

Tumor Necrosis Factor α (TNFα) is best known as a mediator of inflammation and immunity, and also plays important roles in tumor biology. However, the role of TNFα in tumor biology is complex and not completely understood. In a human melanoma cell line, M2, and a lung carcinoma cell line, A549, TNFα up-regulates prion protein (PrP) level, and promotes tumor cell migration in a PrP dependent manner. Silencing PRNP abrogates TNFα induced tumor cell migration; this phenotype is reversed when PRNP is re-introduced. Treatment with TNFα activates nuclear factor kappa B (NF-κB) signaling, which then mitigates autophagy by reducing the expression of Forkhead Box P3 (FOXP3). Down regulation of FOXP3 reduces the transcription of synaptosome associated protein 29 (SNAP29), which is essential in the fusion of autophagosome and lysosome creating autolysosome. FOXP3 being a bona fide transcription factor for SNAP29 is confirmed in a promoter binding assay. Accordingly, silencing SNAP29 in these cell lines also up-regulates PrP, and promotes tumor cell migration without TNFα treatment. But, when SNAP29 or FOXP3 is silenced in these cells, they are no longer respond to TNFα. Thus, a reduction in autophagy is the underlying mechanism by which expression of PrP is up-regulated, and tumor cell migration is enhanced upon TNFα treatment. Disrupting the TNFα-NF-κB-FOXP3-SNAP29 signaling axis may provide a therapeutic approach to mitigate tumor cell migration.

Melanoma migration is promoted by prion protein via Akt-hsp27 signaling axis.

Patients with metastatic melanoma have a poorer prognosis. Prion protein (PrP) in melanoma is known to play an important role in cancer cell migration and invasion by interacting with filamin A (FLNa), a cytolinker protein. To investigate if PrP may contribute to cancer cell mobility independent of its binding to FLNa, we knocked out PRNP in M2 melanoma cell, which lacked FLNa expression. We found that deletion of PRNP in M2 significantly reduced its motility. When PRNP was deleted, the level of Akt was decreased. As a consequence, phosphorylation of small heat shock protein (hsp27) was also reduced, which resulted in polymerization of F-actin rendering the cells less migratory. Accordingly, when PrP was re-expressed in PRNP null M2 cells, the mobility of the recurred cells was rescued, so were the expression levels of Akt and phosphorylated hsp27, resulting in a decrease in the polymerization of F-actin. These results revealed that PrP can play a FLNa independent role in cytoskeletal organization and tumor cell migration by modulating Akt-hsp27-F-actin axis.

Prion dimer is heterogenous and is modulated by multiple negative and positive motifs.

The conversion of the normal prion protein (PrP) into a scrapie prion (PrPSc) is incompletely understood. Theoretically, the smallest PrP aggregate is a dimer. Human PrP contains two cysteines at positions 179 (C179) and 214 (C214) enabling disulfide bonding. Here, we report that our recombinant human PrP (r-hPrP) preparations contain 0.2-0.8% dimer, which is linked by either one or two disulfide bonds, connected by C179-C179, C214-C214, or C179-C214. Furthermore, dimerization is regulated by multiple motifs. While residues 36-42 inhibit, residues 90-125, and 195-212 promote dimerization. Mutating individual residue between 36 and 42 enhances dimerization whereas mutating the positively charged residues within 95-115, or the negatively charged residues within 195-212 prevent dimerization. Although deletion of the entire octapeptide-repeat (5OR) region prevents dimerization, mutating the histidines within the 5OR enhances dimerization. In addition, we found that two out of three brain lysates from patients with inherited prion disease had more PrP dimers than controls. Thus, PrP dimerization may contribute to prion diseases.

CD2-Associated Protein Contributes to Hepatitis C, Virus Propagation and Steatosis by Disrupting Insulin Signaling.

Chronic hepatitis C virus (HCV) infection can result in steatosis, a condition displaying aberrant accumulation of neutral lipid vesicles, the component of lipid droplets (LDs), which are essential for HCV assembly. However, the interplay between HCV infection and steatosis remains unclear. Here, we show that HCV-infected cells have higher levels of CD2-associated protein (CD2AP), which plays two distinct, yet tightly linked, roles in HCV pathogenesis: Elevated CD2AP binds to nonstructural protein 5A (NS5A) and participates in the transport of NS5A to LDs to facilitate viral assembly; Up-regulated CD2AP also interacts with casitas B-lineage lymphoma (b) (Cbl/Cbl-b) E3 ligases to degrade insulin receptor substrate 1 (IRS1), which, in turn, disrupts insulin signaling and increases LD accumulation through the IRS1/protein kinase B (Akt)/adenosine monophosphate-activated protein kinase (AMPK)/hormone-sensitive lipase (HSL) signaling axis to accommodate viral assembly. In the HCV-infected mouse model, CD2AP expression is up-regulated during the chronic infection stage and this up-regulation correlates well with liver steatosis. Importantly, CD2AP up-regulation was also detected in HCV-infected human liver biopsies showing steatosis compared to non-HCV-infected controls. Conclusion: CD2AP is indicated as a protein up-regulated by HCV infection, which, in turn, stimulates HCV propagation and steatosis by disrupting insulin signaling; targeting CD2AP may offer an opportunity for alleviating HCV infection and its associated liver pathology. (Hepatology 2018;XX:XXX-XXX.).

Prion protein is required for tumor necrosis factor α (TNFα)-triggered nuclear factor κB (NF-κB) signaling and cytokine production.

The expression of normal cellular prion protein (PrP) is required for the pathogenesis of prion diseases. However, the physiological functions of PrP remain ambiguous. Here, we identified PrP as being critical for tumor necrosis factor (TNF) α-triggered signaling in a human melanoma cell line, M2, and a pancreatic ductal cell adenocarcinoma cell line, BxPC-3. In M2 cells, TNFα up-regulates the expression of p-IκB-kinase α/ß (p-IKKα/ß), p-p65, and p-JNK, but down-regulates the IκBα protein, all of which are downstream signaling intermediates in the TNF receptor signaling cascade. When PRNP is deleted in M2 cells, the effects of TNFα are no longer detectable. More importantly, p-p65 and p-JNK responses are restored when PRNP is reintroduced into the PRNP null cells. TNFα also activates NF-κB and increases TNFα production in wild-type M2 cells, but not in PrP-null M2 cells. Similar results are obtained in the BxPC-3 cells. Moreover, TNFα activation of NF-κB requires ubiquitination of receptor-interacting serine/threonine kinase 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). TNFα treatment increases the binding between PrP and the deubiquitinase tumor suppressor cylindromatosis (CYLD), in these treated cells, binding of CYLD to RIP1 and TRAF2 is reduced. We conclude that PrP traps CYLD, preventing it from binding and deubiquitinating RIP1 and TRAF2. Our findings reveal that PrP enhances the responses to TNFα, promoting proinflammatory cytokine production, which may contribute to inflammation and tumorigenesis.

Novel strain properties distinguishing sporadic prion diseases sharing prion protein genotype and prion type.

In most human sporadic prion diseases the phenotype is consistently associated with specific pairings of the genotype at codon 129 of the prion protein gene and conformational properties of the scrapie PrP (PrPSc) grossly identified types 1 and 2. This association suggests that the 129 genotype favours the selection of a distinct strain that in turn determines the phenotype. However, this mechanism cannot play a role in the phenotype determination of sporadic fatal insomnia (sFI) and a subtype of sporadic Creutzfeldt-Jakob disease (sCJD) identified as sCJDMM2, which share 129 MM genotype and PrPSc type 2 but are associated with quite distinct phenotypes. Our detailed comparative study of the PrPSc conformers has revealed major differences between the two diseases, which preferentially involve the PrPSc component that is sensitive to digestion with proteases (senPrPSc) and to a lesser extent the resistant component (resPrPSc). We conclude that these variations are consistent with two distinct strains in sFI and sCJDMM2, and that the rarer sFI is the result of a variant strain selection pathway that might be favoured by a different brain site of initial PrPSc formation in the two diseases.

Polyglutamine Tract Expansion Increases S-Nitrosylation of Huntingtin and Ataxin-1.

Expansion of the polyglutamine (polyQ) tract in the huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited movement disorder linked to neurodegeneration in the striatum and cortex. S-nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-nitrosylation affects the biology of this protein in vivo.

Cellular Prion Protein Mediates Pancreatic Cancer Cell Survival and Invasion through Association with and Enhanced Signaling of Notch1.

Up-regulation of human prion protein (PrP) in patients with pancreatic ductal adenocarcinoma (PDAC) is associated with a poor prognosis. However, the underlying molecular mechanism of PrP-mediated tumorigenesis is not completely understood. In this study, we found that PDAC cell lines can be divided into either PrP high expresser or PrP low expresser. In addition to filamin A (FLNA), PrP interacts with Notch1, forming a PrP/FLNA/Notch1 complex. Silencing PrP in high-expresser cells decreases Notch1 expression and Notch1 signaling. These cells exhibited decreased proliferation, xenograft growth, and tumor invasion but show increased tumor apoptosis. These phenotypes were rescued by ectopically expressed and activated Notch1. By contrast, overexpression of PrP in low expressers increases Notch1 expression and signaling, enhances proliferation, and increases tumor invasion and xenograft growth that can be blocked by a Notch inhibitor. Our data further suggest that PrP increases Notch1 stability likely through suppression of Notch proteosome degradation. Additionally, we found that targeting PrP combined with anti-Notch is much more effective than singularly targeted therapy in retarding PDAC growth. Finally, we show that coexpression of PrP and Notch1 confers an even poorer prognosis than PrP expression alone. Taken together, our results have unraveled a novel molecular pathway driven by interactions between PrP and Notch1 in the progression of PDAC, supporting a critical tumor-promoting role of Notch1 in PrP-expressing PDAC tumors.

Glycan-deficient PrP stimulates VEGFR2 signaling via glycosaminoglycan.

Whether the two N-linked glycans are important in prion, PrP, biology is unresolved. In Chinese hamster ovary (CHO) cells, the two glycans are clearly not important in the cell surface expression of transfected human PrP. Compared to fully-glycosylated PrP, glycan-deficient PrP preferentially partitions to lipid raft. In CHO cells glycan-deficient PrP also interacts with glycosaminoglycan (GAG) and vascular endothelial growth factor receptor 2 (VEGFR2), resulting in VEGFR2 activation and enhanced Akt phosphorylation. Accordingly, CHO cells expressing glycan-deficient PrP lacking the GAG binding motif or cells treated with heparinase to remove GAG show diminished Akt signaling. Being in lipid raft is critical, chimeric glycan-deficient PrP with CD4 transmembrane and cytoplasmic domains is absent in lipid raft and does not activate Akt signaling. CHO cells bearing glycan-deficient PrP also exhibit enhanced cellular adhesion and migration. Based on these findings, we propose a model in which glycan-deficient PrP, GAG, and VEGFR2 interact, activating VEGFR2 and resulting in changes in cellular behavior.

Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility.

The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. "Knocking out" PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility.

Anti-lubricin monoclonal antibodies created using lubricin-knockout mice immunodetect lubricin in several species and in patients with healthy and diseased joints.

Lubricin, encoded by the gene PRG4, is the principal lubricant in articulating joints. We immunized mice genetically deficient for lubricin (Prg4-/-) with purified human lubricin, and generated several mAbs. We determined each mAb's binding epitope, sensitivity, and specificity using biologic samples and recombinant lubricin sub-domains, and we also developed a competition ELISA assay to measure lubricin in synovial fluid and blood. We found the mAbs all recognized epitopes containing O-linked oligosaccharides conjugated to the peptide motif KEPAPTTT. By western blot, the mAbs detected lubricin in 1 µl of synovial fluid from several animal species, including human. The mAbs were specific for lubricin since they did not cross-react with other synovial fluid constituents from patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), who genetically lack this protein. The competition ELISA detected lubricin in blood samples from healthy individuals but not from patients with CACP, indicating blood can be used in a diagnostic test for patients suspected of having CACP. Lubricin epitopes in blood do not represent degradation fragments from synovial fluid. Therefore, although blood lubricin levels did not differentiate patients with inflammatory joint disease from healthy controls, epitope-specific anti-lubricin mAbs could be useful for monitoring disease activity in synovial fluid.

A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells.

Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells (HPDC).

Small protease sensitive oligomers of PrPSc in distinct human prions determine conversion rate of PrP(C).

The mammalian prions replicate by converting cellular prion protein (PrP(C)) into pathogenic conformational isoform (PrP(Sc)). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP(Sc) on conversion of PrP(C) in vitro using PrP(Sc) seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP(Sc). The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP(Sc). The tight correlation between conversion potency of small oligomers of human sPrP(Sc) observed in vitro and duration of the disease suggests that sPrP(Sc) conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.

Protease-sensitive conformers in broad spectrum of distinct PrPSc structures in sporadic Creutzfeldt-Jakob disease are indicator of progression rate.

The origin, range, and structure of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD), are largely unknown. To investigate the molecular mechanism responsible for the broad phenotypic variability of sCJD, we analyzed the conformational characteristics of protease-sensitive and protease-resistant fractions of the pathogenic prion protein (PrP(Sc)) using novel conformational methods derived from a conformation-dependent immunoassay (CDI). In 46 brains of patients homozygous for polymorphisms in the PRNP gene and exhibiting either Type 1 or Type 2 western blot pattern of the PrP(Sc), we identified an extensive array of PrP(Sc) structures that differ in protease sensitivity, display of critical domains, and conformational stability. Surprisingly, in sCJD cases homozygous for methionine or valine at codon 129 of the PRNP gene, the concentration and stability of protease-sensitive conformers of PrP(Sc) correlated with progression rate of the disease. These data indicate that sCJD brains exhibit a wide spectrum of PrP(Sc) structural states, and accordingly argue for a broad spectrum of prion strains coding for different phenotypes. The link between disease duration, levels, and stability of protease-sensitive conformers of PrP(Sc) suggests that these conformers play an important role in the pathogenesis of sCJD.