Cytosolic prion protein is the predominant anti-Bax prion protein form: exclusion of transmembrane and secreted prion protein forms in the anti-Bax function

Prion protein (PrP) prevents Bax-mediated cell death by inhibiting the initial Bax conformational change that converts cytosolic Bax into a pro-apoptotic protein. PrP is mostly a glycophosphatidylinositol-anchored cell surface protein but it is also retrotranslocated into cytosolic PrP (CyPrP) or can become a type 1 or type 2 transmembrane protein. To determine the form and subcellular location of the PrP that has anti-Bax function, we co-expressed various Syrian hamster PrP (SHaPrP) mutants that favour specific PrP topologies and subcellular localization with N-terminally green fluorescent protein tagged pro-apoptotic Bax (EGFP-Bax) in MCF-7 cells and primary human neurons. Mutants that generate both CyPrP and secreted PrP ((Sec)PrP) or only CyPrP have anti-Bax activity. Mutants that produce (Ctm)PrP or (Ntm)PrP lose the anti-Bax activity, despite their ability to also make (Sec)PrP. Transmembrane-generating mutants do not produce CyPrP and both normal and cognate mutant forms of CyPrP rescue against the loss of anti-Bax activity. (Sec)PrP-generating constructs also produce non-membrane attached (Sec)PrP. However, this form of PrP has minimal anti-Bax activity. We conclude that CyPrP is the predominant form of PrP with anti-Bax function. These results imply that the retrotranslocation of PrP encompasses a survival function and is not merely a pathway for the proteasomal degradation of misfolded protein.     

Loss of Anti-Bax Function in Gerstmann-Str?ussler-Scheinker Syndrome-Associated Prion Protein Mutants

Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD)-associated prion protein (PrP) mutants that are unable to generate cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS)-associated PrP mutants. These PrP mutants contained their respective methionine (^M) or valine (^V) at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S^V, D202N^V, P102L^V and Q217R^V retained, whereas the P102L^M, P105L^V, Y145stop^M and Q212P^M PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L^V, none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.     

How Does Domain Replacement Affect Fibril Formation of the Rabbit/Human Prion Proteins

Background

It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.

Methodology/Principal Findings

As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.

Conclusions/Significance

We demonstrate for the first time that the domains beyond PrP-H2H3 (β-strand 1, α-helix 1, and β-strand 2) have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different effects on fibrillization of PrPs from different species.     

PrP mutants with different numbers of octarepeat sequences are more susceptible to the oxidative stress

One of the physiological functions of cellular prion protein(PrP C )is believed to work as a cellular resistance to oxidative stress,in which the octarepeats region within PrP plays an important role.However,the detailed mechanism is less clear.In this study,the expressing plasmids of wild-type PrP (PrP-PG5)and various PrP mutants containing 0(PrP-PG0),9(PrP-PG9)and 12(PrP-PG12)octarepeats were generated and PrP proteins were expressed both in E.coli and in mammalian cells.Protein aggregation and formation of carbonyl groups were clearly seen in the recombinant PrPs expressed from E.coli after treatment of H2O2.MTT and trypan blue staining assays revealed that the cells expressing the mutated PrPs within octarepeats are less viable than the cells expressing wild-type PrP.Statistically significant high levels of intracellular free radicals and low levels of glutathione peroxidase were observed in the cells transfected with plasmids containing deleted or inserted octarepeats.Remarkably more productions of carbonyl groups were detected in the cells expressing PrPs with deleted and inserted octarepeats after exposing to H2O2.Furthermore,cells expressing wild-type PrP showed stronger resistant activity to the challenge of H2O2 at certain extent than the mutated PrPs and mock. These data provided the evidences that the octarepeats number within PrP is critical for maintaining its activity of antioxidation.Loss of its protective function against oxidative stress may be one of the possible pathways for the mutated PrPs to involve in the pathogenesis of familial Creutzfeldt-Jacob diseases.     

Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein

Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrP^Sc of the host-encoded prion protein (PrP^c). A profound conformational change of PrP^c underlies formation of PrP^Sc and prion propagation involves conversion of PrP^c substrate by direct interaction with PrP^Sc template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrP^C into PrP^Sc. To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrP^Sc recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated.     

Salt bridges in prion proteins are necessary for high-affinity binding to the monoclonal antibody T2

We studied the role of the 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) in helix 1 of mouse prion protein (PrP) on the formation of the complex between PrP and the monoclonal antibody T2. We introduced 6 charge-changing mutations to the amino acid residues associated with the salt bridges. Analysis of the circular dichroism spectra of the mutant PrPs showed that the salt bridge mutations did not change the secondary structures. We analyzed the kinetics of the association and dissociation of the PrPs with the T2 antibody. The results showed that the association kinetics were not significantly different among the variants except Arg150Lys, while the dissociation rate of the neutralized-charge variants was 2 orders of magnitude higher than that of the wild type. These results indicate that salt bridges make the interaction of PrP with T2 tighter by slowing down dissociation.     

A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP^C into pathogenic PrP^Sc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.     

Molecular interaction of TPPP with PrP antagonized the CytoPrP-induced disruption of microtubule structures and cytotoxicity

Background

Tubulin polymerization promoting protein/p25 (TPPP/p25), known as a microtubule-associated protein (MAP), is a brain-specific unstructured protein with a physiological function of stabilizing cellular microtubular ultrastructures. Whether TPPP involves in the normal functions of PrP or the pathogenesis of prion disease remains unknown. Here, we proposed the data that TPPP formed molecular complex with PrP. We also investigated its influence on the aggregation of PrP and fibrillization of PrP106–126 in vitro, its antagonization against the disruption of microtubule structures and cytotoxicity of cytosolic PrP in cells, and its alternation in the brains of scrapie-infected experimental hamsters.

Methodology/Principal Findings

Using pull-down and immunoprecipitation assays, distinct molecular interaction between TPPP and PrP were identified and the segment of TPPP spanning residues 100–219 and the segment of PrP spanning residues 106–126 were mapped as the regions responsible for protein interaction. Sedimentation experiments found that TPPP increased the aggregation of full-length recombinant PrP (PrP23–231) in vitro. Transmission electron microscopy and Thioflavin T (ThT) assays showed that TPPP enhanced fibril formation of synthetic peptide PrP106–126 in vitro. Expression of TPPP in the cultured cells did not obviously change the microtubule networks observed by a tubulin-specific immunofluorescent assay and cell growth features measured by CCK8 tests, but significantly antagonized the disruption of microtubule structures and rescued the cytotoxicity caused by the accumulation of cytosolic PrP (CytoPrP). Furthermore, Western blots identified that the levels of the endogenous TPPP in the brains of scrapie-infected experimental hamsters were significantly reduced.

Conclusion/Significance

Those data highlight TPPP may work as a protective factor for cells against the damage effects of the accumulation of abnormal forms of PrPs, besides its function as an agent for dynamic stabilization of microtubular ultrastructures.     

Removal of the glycosylation of prion protein provokes apoptosis in SF126

Although the function of cellular prion protein (PrPc) and the pathogenesis of prion diseases have been widely described, the mechanisms are not fully clarified. In this study, increases of the portion of non-glycosylated prion protein deposited in the hamster brains infected with scrapie strain 263K were described. To elucidate the pathological role of glycosylation profile of PrP, wild type human PrP (HuPrP) and two genetic engineering generated non-glycosylated PrP mutants (N181Q/N197Q and T183A/T199A) were transiently expressed in human astrocytoma cell line SF126. The results revealed that expressions of non-glycosylated PrP induced significantly more apoptosis cells than that of wild type PrP. It illustrated that Bcl-2 proteins might be involved in the apoptosis pathway of non-glycosylated PrPs. Our data highlights that removal of glycosylation of prion protein provokes cells apoptosis.     

Polo-like kinase 3 (PLK3) mediates the clearance of the accumulated PrP mutants transiently expressed in cultured cells and pathogenic PrPSc in prion infected cell line via protein interaction

Polo-like kinases (PLKs) family has long been known to be critical for cell cycle and recent studies have pointed to new dimensions of PLKs function in the nervous system. Our previous study has verified that the levels of PLK3 in the brain are severely downregulated in prion-related diseases. However, the associations of PLKs with prion protein remain unclear. In the present study, we confirmed that PrP protein constitutively interacts with PLK3 as determined by both in vitro and in vivo assays. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. Overexpression of PLK3 did not affect the level of wild-type PrP, but significantly decreased the levels of the mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed to be independent of its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrPs. Moreover, PLK3 overexpression in a scrapie infected cell line caused notable reduce of PrP^Sc level in a dose-dependent manner, but had minimal effect on the expression of PrP^C in its normal partner cell line. Our findings here confirmed the molecular interaction between PLK3 and PrP and outlined the regulatory activity of PLK3 on the degradation of abnormal PrPs, even its pathogenic isoform PrP^Sc. We, therefore, assume that the recovery of PLK3 in the early stage of prion infection may be helpful to prevent the toxic accumulation of PrP^Sc in the brain tissues.     

Prion protein helix1 promotes aggregation but is not converted into beta-sheet

Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).     

Conserved Roles of the Prion Protein Domains on Subcellular Localization and Cell-Cell Adhesion

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP’s essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP’s ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.     

The crystal structure of the globular domain of sheep prion protein

The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.     

A more robust version of the Arginine 210-switched mutant in subunit a of the Escherichia coli ATP synthase

Previous work has shown that the essential R210 of subunit a in the Escherichia coli ATP synthase can be switched with a conserved glutamine Q252 with retention of a moderate level of function, that a third mutation P204T enhances this function, and that the arginine Q252R can be replaced by lysine without total loss of activity. In this study, the roles of P204T and R210Q were examined. It was concluded that the threonine in P204T is not directly involved in function since its replacement by alanine did not significantly affect growth properties. Similarly, it was concluded that the glutamine in R210Q is not directly involved with function since replacement by glycine results in significantly enhanced function. Not only did the rate of ATP-driven proton translocation increase, but also the sensitivity of ATP hydrolysis to inhibition by N,N′-dicyclohexylcarbodiimide (DCCD) rose to more than 50%. Finally, mutations at position E219, a residue near the proton pathway, were used to test whether the Arginine-switched mutant uses the normal proton pathway. In a wild type background, the E219K mutant was confirmed to have greater function than the E219Q mutant, as has been shown previously. This same unusual result was observed in the triple mutant background, P204T/R210Q/Q252R, suggesting that the Arginine-switched mutants are using the normal proton pathway from the periplasm.     

Oxidative impairment in scrapie-infected mice is associated with brain metals perturbations and altered antioxidant activities

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)). PrP(C) binds copper, has superoxide dismutase (SOD)-like activity in vitro, and its expression aids in the cellular response to oxidative stress. However, the interplay between PrPs (PrP(C), PrP(Sc) and possibly other abnormal species), copper, anti-oxidation activity and pathogenesis of prion diseases remain unclear. In this study, we reported dramatic depression of SOD-like activity by the affinity-purified PrPs from scrapie-infected brains, and together with significant reduction of Cu/Zn-SOD activity, correlates with significant perturbations in the divalent metals contents. We also detected elevated levels of nitric oxide and superoxide in the infected brains, which could be escalating the oxidative modification of cellular proteins, reducing gluathione peroxidase activity and increasing the levels of lipid peroxidation markers. Taken together, our results suggest that brain metal imbalances, especially copper, in scrapie infection is likely to affect the anti-oxidation functions of PrP and SODs, which, together with other cellular dysfunctions, predispose the brains to oxidative impairment and eventual degeneration. To our knowledge, this is the first study documenting a physiological connection between brain metals imbalances, the anti-oxidation function of PrP, and aberrations in the cellular responses to oxidative stress, in scrapie infection.     

Molecular dynamics simulations of human prion protein: importance of correct treatment of electrostatic interactions.

Molecular dynamics simulations have been used to investigate the dynamical and structural behavior of a homology model of human prion protein HuPrP(90-230) generated from the NMR structure of the Syrian hamster prion protein ShPrP(90-231) and of ShPrP(<90-231) itself. These PrPs have a large number of charged residues on the protein surface. At the simulation pH 7, HuPrP(90-230) has a net charge of -1 eu from 15 positively and 14 negatively charged residues. Simulations for both PrPs, using the AMBER94 force field in a periodic box model with explicit water molecules, showed high sensitivity to the correct treatment of the electrostatic interactions. Highly unstable behavior of the structured region of the PrPs (127-230) was found using the truncation method, and stable trajectories could be achieved only by including all the long-range electrostatic interactions using the particle mesh Ewald (PME) method. The instability using the truncation method could not be reduced by adding sodium and chloride ions nor by replacing some of the sodium ions with calcium ions. The PME simulations showed, in accordance with NMR experiments with ShPrP and mouse PrP, a flexibly disordered N-terminal part, PrP(90-126), and a structured C-terminal part, PrP(127-230), which includes three alpha-helices and a short antiparallel beta-strand. The simulations showed some tendency for the highly conserved hydrophobic segment PrP(112-131) to adopt an alpha-helical conformation and for helix C to split at residues 212-213, a known disease-associated mutation site (Q212P). Three highly occupied salt bridges could be identified (E146/D144<-->R208, R164<-->D178, and R156<-->E196) which appear to be important for the stability of PrP by linking the stable main structured core (helices B and C) with the more flexible structured part (helix A and strands A and B). Two of these salt bridges involve disease-associated mutations (R208H and D178N). Decreased PrP stability shown by protein unfolding experiments on mutants of these residues and guanidinium chloride or temperature-induced unfolding studies indicating reduced stability at low pH are consistent with stabilization by salt bridges. The fact that electrostatic interactions, in general, and salt bridges, in particular, appear to play an important role in PrP stability has implications for PrP structure and stability at different pHs it may encounter physiologically during normal or abnormal recycling from the pH neutral membrane surface into endosomes or lysomes (acidic pHs) or in NMR experiments (5.2 for ShPrP and 4.5 for mouse PrP).     

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.     

Cooperative binding of dominant-negative prion protein to kringle domains

Conversion of the cellular prion protein (PrP(C)) to the pathogenic isoform (PrP(Sc)) is a major biochemical alteration in the progression of prion disease. This conversion process is thought to require interaction between PrP(C) and an as yet unidentified auxiliary factor, provisionally designated protein X. In searching for protein X, we screened a phage display cDNA expression library constructed from prion-infected neuroblastoma (ScN2a) cells and identified a kringle protein domain using full-length recombinant mouse PrP (recMoPrP(23-231), hereafter recMoPrP) expressing a dominant-negative mutation at codon 218 (recMoPrP(Q218K)). In vitro binding analysis using ELISA verified specific interaction of recMoPrP to kringle domains (K(1+2+3)) with higher binding by recMoPrP(Q218K) than by full-length recMoPrP without the mutation. This interaction was confirmed by competitive binding analysis, in which the addition of either a specific anti-kringle antibody or L-lysine abolished the interaction. Biochemical studies of the interactions between K(1+2+3) and various concentrations of both recMoPrP molecules demonstrated binding in a dose-dependent manner. A Hill plot analysis of the data indicates positive cooperative binding of both recMoPrP(Q218K) and recMoPrP to K(1+2+3) with stronger binding by recMoPrP(Q218K). Using full-length and an N-terminally truncated MoPrP(89-231), we demonstrate that N-terminal sequences enable PrP to bind strongly to K(1+2+3). Further characterization with truncated MoPrP(89-231) refolded in different conformations revealed that both alpha-helical and beta-sheet conformations bind to K(1+2+3). Our data demonstrate specific, high-affinity binding of a dominant-negative PrP as well as binding of other PrPs to K(1+2+3). The relevance of such interactions during prion pathogenesis remains to be established.     

Disulfide bond as a structural determinant of prion protein membrane insertion

Conversion of the normal soluble form of prion protein, PrP (PrP^C), to proteinase K-resistant form (PrP^Sc) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from α-helix to β-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111–135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.     

Cellular prion protein inhibits proapoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells

Prion protein (PrP) prevents Bcl-2-associated protein X (Bax)-mediated cell death, but the step at which PrP inhibits is not known. We first show that PrP is very specific for Bax and cannot prevent Bak (Bcl-2 antagonist killer 1)-, tBid-, staurosporine- or thapsigargin-mediated cell death. As Bax activation involves Bax conformational change, mitochondrial translocation, cytochrome c release and caspase activation, we investigated which of these events was inhibited by PrP. PrP inhibits Bax conformational change, cytochrome c release and cell death in human primary neurons and MCF-7 cells. Serum deprivation-induced Bax conformational change is more rapid in PrP-null cells. PrP does not prevent active caspase-mediated cell death. PrP does not colocalize with Bax in normal or apoptotic primary neurons and cannot prevent Bax-mediated cytochrome c release in a mitochondrial cell-free system. We conclude that PrP protects against Bax-mediated cell death by preventing the Bax proapoptotic conformational change that occurs initially in Bax activation.