PrP cooperates with STI1 to regulate SOD activity in PrP-deficient neuronal cell line

Cellular prion protein (PrP(C)) plays anti-apoptotic and anti-oxidative roles in apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line. The octapeptide repeat region (OR) and N-terminal half of the hydrophobic region (HR) of PrP(C) are indispensable for PrP(C) activity, but the mechanisms remain unclear. In the present study, elucidation of the mechanisms by which PrP(C) elicits the anti-oxidative activities was facilitated by evidence of stress-inducible protein 1 (STI1) mediating PrP(C)-dependent superoxide dismutase (SOD) activation. Immunoprecipitation revealed that PrP(C) was associated with STI1. The inhibitory peptides against PrP(C)-STI1 binding [STI1 pep.1 and PrP(113-132)] indicated toxic activity in PrP(C)-expressing cells by inhibiting SOD activity but not in Prnp(-/-) cells. Furthermore, OR and N-terminal half of the HR were required for the inhibitory effect of PrP(113-132) but not STI1 pep.1. These data are consistent with results established with a model where OR and N-terminal half of the HR mediate the action of STI1 upon cell survival and upregulation of SOD activity.     

Fusion of Doppel to octapeptide repeat and N-terminal half of hydrophobic region of prion protein confers resistance to serum deprivation

Our previous studies have shown an essential role played by the octapeptide repeat region (OR) and the N-terminal half of hydrophobic region (HR) in the anti-apoptotic activity of prion protein (PrP). As PrP-like protein Doppel (Dpl), which structurally resembles an N-terminally truncated PrP, did not show any anti-apoptotic activity, we examined apoptosis of HpL3-4 cells expressing Dpl fused to various lengths of the N-terminal region of PrP to investigate whether the PrP/Dpl fusion proteins retain anti-apoptotic function. HpL3-4 cells expressing Dpl fused to PrP(1-124) with the OR and N-terminal half of HR of PrP showed anti-apoptotic function, whereas Dpl fused to PrP(1-95) with OR did not rescue cells from apoptotic cell death induced by serum deprivation. These results indicate that the OR and N-terminal half of HR of PrP retains anti-apoptotic activity similar to full-length PrP.     

Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line

Previous studies have reported a neuroprotective role for cellular prion protein (PrP(C)) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrP(C) prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrP(C) or PrP(C) lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrP(C) enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrP(C) protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrP(C) plays an essential role in regulating apoptosis and SOD activity.     

Neurotoxic prion protein (PrP) fragment 106-126 requires the N-terminal half of the hydrophobic region of PrP in the PrP-deficient neuronal cell line

The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.     

Octapeptide repeat region of prion protein (PrP) is required at an early stage for production of abnormal prion protein in PrP-deficient neuronal cell line

An abnormal isoform of prion protein (PrP^Sc), which is composed of the same amino acids as cellular PrP (PrP^C) and has proteinase K (PK)-resistance, hypothetically converts PrP^C into PrP^Sc. To investigate the region important for PrP^Sc production, we examined the levels of PrP^Sc in PrP gene-deficient cells (HpL3-4) expressing PrP^C deleted of various regions including the octapeptide repeat region (OR) or hydrophobic region (HR). After Chandler or Obihiro prion infection, PrP^Sc was produced in HpL3-4 cells expressing wild-type PrP^C or PrP^C deleted of HR at an early stage and further reduced to below the detectable level, whereas cells expressing PrP^C deleted of OR showed no PrP^Sc production. The results suggest that OR of PrP^C is required for the early step of efficient PrP^Sc production.     

Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique

We analyzed the pH-induced mobility changes in moPrP(C) alpha-helix and beta-sheets by cysteine-scanning site-directed spin labeling (SDSL) with ESR. Nine amino acid residues of alpha-helix1 (H1, codon 143-151), four amino acid residues of beta-sheet1 (S1, codon 127-130), and four amino acid residues of beta-sheet2 (S2, codon 160-163) were substituted for by cysteine residues. These recombinant mouse PrP(C) (moPrP(C)) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/deltaH of the central (14N hyperfine) component (M(I) = 0) in the ESR spectrum of spin-labeled moPrP(C) was measured as a mobility parameter of nitroxide residues (R1). The mobilities of E145R1 and Y149R1 at pH 7.4, which was identified as a tertiary contact site by a previous NMR study of moPrP, were lower than those of D143R1, R147R1, and R150R1 reported on the helix surface. Thus, the mobility in the H1 region in the neutral solution was observed with the periodicity associated with a helical structure. On the other hand, the values in the S2 region, known to be located in the buried side, were lower than those in the S1 region located in the surface side. These results indicated that the mobility parameter of the nitroxide label was well correlated with the 3D structure of moPrP. Furthermore, the present study clearly demonstrated three pH-sensitive sites in moPrP, i.e., (1) the N-terminal tertiary contact site of H1, (2) the C-terminal end of H1, and (3) the S2 region. In particular, among these pH-sensitive sites, the N-terminal tertiary contact region of H1 was found to be the most pH-sensitive one and was easily converted to a flexible structure by a slight decrease of pH in the solution. These data provided molecular evidence to explain the cellular mechanism for conversion from PrP(C) to PrP(Sc) in acidic organelles such as the endosome.     

The N-terminal, polybasic region is critical for prion protein neuroprotective activity

Several lines of evidence suggest that the normal form of the prion protein, PrP(C), exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrP(C) to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (Δ32-134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (Δ23-31, Δ23-111, and Δ23-134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although Δ23-31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrP(C) neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases.     

Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions

Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrPΔOR)/Prnp(0/0) mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrPΔOR, or PrP(Sc)ΔOR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrP(Sc)ΔOR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrP(Sc)ΔOR, including the pre-OR residues 23-50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.     

Cell cycle and activation of CK2

Microtubule associated protein tau is considered to play roles in some types of human transmissible spongiform encephalopathies (TSE). In this study, the full-length and several truncated human tau proteins were expressed from E. coli and purified. Using GST pull down, co-immunoprecipitation assay and tau-coated ELISA, the molecular interaction between tau protein and PrP was confirmed in the context of the full-length human tau. The N terminus (amino acids 1–91) and tandem repeats region (amino acids 186–283) of tau protein were responsible for the interaction with PrP. The octapeptide repeats within PrP directly affected the binding activity of PrP with tau. GSS-related mutant PrP102L and fCJD- related mutants with two and seven extra octarepeats showed more active binding capacity with tau than wild-type PrP. The molecular interactions between PrP and tau protein highlight a potential role of tau in the biological function of PrP and the pathogenesis of TSE.     

Phosphorylation of Kruppel-like factor 5 (KLF5/IKLF) at the CBP interaction region enhances its transactivation function

The Kruppel-like factor 5 (KLF5/IKLF) belongs to the Kruppel family of genes which bind GC-rich DNA elements and activate or repress their target genes in a promoter context and/or cellular environment-dependent manner. In the present study, we used the Gal4 fusion assay system to characterize the mechanism of transactivation by KLF5. We demonstrated that the transactivation function of KLF5 was enhanced by CREB-binding protein (CBP) and blocked by wild-type but not mutant E1A. Over expression of CBP reversed the inhibition effect of E1A. With various lengths of KLF5 fusion protein, the transactivation functions were localized to 156 amino acid residues at the N-terminal region and 133 amino acid residues adjacent to the Zn finger motif. We mapped the CBP and KLF5 interaction domain to the N-terminal region of CBP (amino acids 1–232) and the N-terminal region of KLF5 (amino acids 1–238) where one of the activation functions resides. The histone acetyltransferase (HAT) activity of CBP does not play a role in the transactivation function of KLF5 nor does it acetylate KLF5 in vitro. However, phosphorylation is important in KLF5 transactivation activity. Inhibition of protein kinase activity by H7 or calphostin C blocked both full-length and N-terminal fragment (amino acids 1–238) KLF5 activities. Mutation at a potential protein kinase C phosphorylation site within the CBP interaction domain of KLF5 reduces its transactivation function. Furthermore, using the GST pull-down approach, we showed that phosphorylation of KLF5 enhances its interaction with CBP. The results of the present study provide a mechanism for KLF5 transactivation function.     

Cloning and expression of a prion protein (PrP) gene from Korean bovine (Bos taurus coreanae) and production of rabbit anti-bovine PrP antibody

A PrP gene, from a Korean bovine, exhibiting a nonsense and a missense polymorphism respectively at nucleotides 576 and 652 has been cloned. The latter resulted in Glu to Lys substitution at amino acid residue 218. After expression and purification of the recombinant bovine PrP (recBoPrP) with Glu218Lys substitution, a polyclonal antibody against this protein was raised. ELISA and Western blot analysis suggested that the recBoPrP obtained in this study had a unique conformation not presented in native PrP(C), and the polyclonal antibody recognized PrP in a conformation dependent manner. These reagents will be valuable tools for studying PrP conformation.     

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.     

Activation of phosphatidylinositol 3-kinase by cellular prion protein and its role in cell survival

The cellular prion protein (PrP(C)) is thought to be involved in protection against cell death, however the exact cellular mechanisms involved are still controversial. Herein we present data that strongly indicate a functional link between PrP(C) expression and phosphatidylinositol 3-kinase (PI 3-kinase) activation, a protein kinase that plays a pivotal role in cell survival. Both mouse neuroblastoma N2a cells and immortalized murine hippocampal neuronal cell lines expressing wild-type PrP(C) had significantly higher PI 3-kinase activity levels than their respective controls. Moreover, PI 3-kinase activity was found to be elevated in brain lysates from wild-type mice, as compared to prion protein-knockout mice. Recruitment of PI 3-kinase by PrP(C) was shown to contribute to cellular survival toward oxidative stress by using 3-morpholinosydnonimine (SIN-1) and serum deprivation. Moreover, both PI 3-kinase activation and cytoprotection by PrP(C) appeared to rely on copper binding to the N-terminal octapeptide of PrP(C). Thus, we propose a model in which the interaction of copper(II) with the N-terminal domain of PrP(C) enables transduction of a signal to PI 3-kinase; the latter, in turn, mediates downstream regulation of cell survival.     

Mutations in the putative HR-C region of the measles virus F2 glycoprotein modulate syncytium formation

The fusion (F) glycoproteins of measles virus strains Edmonston (MV-Edm) and wtF (MV-wtF) confer distinct cytopathic effects and strengths of hemagglutinin (H) interaction on a recombinant MV-Edm virus. They differ in just two amino acids, V94 and V101 in F-Edm versus M94 and F101 in F-wtF, both of which lie in the relatively uncharacterized F(2) domain. By comparing the sequence of MV F with those of the parainfluenza virus SV5 and Newcastle disease virus (NDV) F proteins, the structures of which are known, we show that MV F(2) also possesses a potential heptad repeat (HR) C domain. In NDV, the N-terminal half of HR-C interacts with HR-A in F(1) while the C-terminal half is induced to kink outward by a central proline residue. We found that this proline is part of an LXP motif conserved in all three viruses. Folding and transport of MV F require this motif to be intact and also require covalent interaction of cysteine residues that probably support the potential HR-A-HR-C interaction. Amino acids 94 and 101, both located in "d" positions of the HR-C helical wheel, lie in the potentially outwardly kinked region. We demonstrate that their effect on MV fusogenicity and glycoprotein interaction is mediated solely by amino acid 94. Substitutions at position 94 with polar or charged amino acids are tolerated poorly or not at all, while changes to smaller and more hydrophilic amino acids are tolerated in both transiently expressed F protein and recombinant virus. MV F V94A and MV F V94G viruses induce extensive syncytium formation and are relatively, or almost completely, resistant to a known inhibitor of MV glycoprotein-induced fusion. We propose that the conformational changes in MV F protein required to expose the fusion peptide involve the C-terminal half of the HR-C helix, specifically amino acid 94.     

Role of N- and C-terminal domains and non-homologous region in co-refolding of Thermotoga maritima β-glucosidase

Thermotoga maritima β-glucosidase consists of three structural regions with 721 amino acids: the N-terminal domain, middle non-homologous region and a C-terminal domain. To investigate the role of these domains in the co-refolding of two fragments into catalytically active form, five sites coding the amino acid residue at 244, 331 in the N-terminal domain, 403 in the non-homologous region, 476 and 521 in the C-terminal domain were selected to split the gene. All the 10 resultant individual fragments were obtained as insoluble inclusion bodies and found to be catalytically inactive. However, the catalytic activity was recovered when the two fragments derived from N-terminal and C-terminal peptides were co-refolded together. It is quite interesting to find that not only the complement polypeptides such as N476/477C but also the truncated combination (N476/522C, amino acid residues from 477 to 521 is truncated) and overlapped combination (N476/245C and N476/404C, amino acid residues from 245 to 476 and from 404 to 476 are overlapped) also gave catalytically active enzymes. Our results showed that folding motifs consisted of the complete N-terminal domain play an important role in the co-refolding of the polypeptides into the catalytically active form.     

N-terminal region of gelsolin induces apoptosis of activated hepatic stellate cells by a caspase-dependent mechanism

Activated hepatic stellate cells (HSCs) are the major source for alteration of extracellular matrix in fibrosis and cirrhosis. Conditioned medium (CM) collected from immortalized human hepatocytes (IHH) have earlier been shown to be responsible for apoptosis of HSCs. In this study, we have shown that antibodies raised against a peptide derived from a linear B-cell epitope in the N-terminal region of gelsolin identified a gelsolin fragment in IHH CM. Analysis of activated stellate cell death by CM collected from Huh7 cells transfected with plasmids encoding gelsolin deletion mutants suggested that the N-terminal half of gelsolin contained sequences which were responsible for stellate cell death. Further analysis determined that this activity was restricted to a region encompassing amino acids 1-70 in the gelsolin sequence; antibody directed to an epitope within this region was able to neutralize stellate cell death. Gelsolin modulation of cell death using this fragment involved upregulation of TRAIL-R1 and TRAIL-R2, and involved caspase 3 activation by extrinsic pathway. The apoptotic activity of N-terminal gelsolin fragments was restricted to activated but not quiescent stellate cells indicating its potential application in therapeutic use as an anti-fibrotic agent. Gelsolin fragments encompassing N-terminal regions in polypeptides of different molecular sizes were detected by N-terminal peptide specific antiserum in IHH CM immunoprecipitated with chronically HCV infected patient sera, suggesting the presence of autoantibodies generated against N-terminal gelsolin fragments in patients with chronic liver disease.     

N-terminal truncation of prion protein affects both formation and conformation of abnormal protease-resistant prion protein generated in vitro

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34-94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.     

The charge structure of helix 1 in the prion protein regulates conversion to pathogenic PrPSc

The prion diseases are transmissible neurodegenerative disorders linked to a pathogenic conformer (PrP(Sc)) of the normal prion protein (PrP(C)). Accumulation of PrP(Sc) occurs via a poorly defined process in which PrP(Sc) complexes with and converts endogenous PrP(C) to nascent PrP(Sc). Recent experiments have focused on the highly charged first alpha helix (H1) of PrP. It has been proposed that two putative asparagine-to-arginine intrahelical salt bridges stabilize H1 in PrP(C) yet form intermolecular ionic bonds with adjacent PrP molecules during conversion of PrP(C) to PrP(Sc) (M. P. Morrissey and E. I. Shakhnovich, Proc. Natl. Acad. Sci. USA 96:11293-11298, 1999). Subsequent work (J. O. Speare et al., J. Biol. Chem. 278:12522-12529, 2003 using a cell-free assay of PrP(Sc) conversion suggested that rather than promoting conversion, the salt bridges stabilize PrP(C) against it. However, the role of individual H1 charges in PrP(Sc) generation has not yet been investigated. To approach this question, we systematically reversed or neutralized each charged residue in H1 and tested the effect on conversion to PrP(Sc) in scrapie-infected murine neuroblastoma (ScN2a) cells. We find that replacements of charged H1 residues with like charges permit conversion, while charge reversals hinder it. Neutralization of charges in the N-terminal (amino acids 143 to 146) but not the C-terminal (amino acids 147 to 151) half of H1 permits conversion, while complete reversal of charge orientation of the putative salt bridges produces a nonconvertible PrP. Circular dichroism spectroscopy studies and confocal microscopy immunofluorescence localization studies indicated that charge substitutions did not alter the secondary structure or cell surface expression of PrP(C). These data support the necessity of specific charge orientations in H1 for a productive PrP(Sc)-PrP(C) complex.     

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.