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The cellular prion protein (PrPC) comprises two domains: a globular C-terminal domain and an unstructured N-terminal domain. Recently, copper has been observed to drive tertiary contact in PrPC, inducing a neuroprotective cis interaction that structurally links the protein's two domains. The location of this interaction on the C terminus overlaps with the sites of human pathogenic mutations and toxic antibody docking. Combined with recent evidence that the N terminus is a toxic effector regulated by the C terminus, there is an emerging consensus that this cis interaction serves a protective role, and that the disruption of this interaction by misfolded PrP oligomers may be a cause of toxicity in prion disease. We demonstrate here that two highly conserved histidines in the C-terminal domain of PrPC are essential for the protein's cis interaction, which helps to protect against neurotoxicity carried out by its N terminus. We show that simultaneous mutation of these histidines drastically weakens the cis interaction and enhances spontaneous cationic currents in cultured cells, the first C-terminal mutant to do so. Whereas previous studies suggested that Cu2+ coordination was localized solely to the protein's N-terminal domain, we find that both domains contribute equatorially coordinated histidine residue side-chains, resulting in a novel bridging interaction. We also find that extra N-terminal histidines in pathological familial mutations involving octarepeat expansions inhibit this interaction by sequestering copper from the C terminus. Our findings further establish a structural basis for PrPC's C-terminal regulation of its otherwise toxic N terminus. Unlabelled Image • The two domains of the cellular prion protein are tethered together by a copper ion. • This tethering is the basis of the protein's neuroprotective cis interaction. • Disruption of this interaction explains the toxicity of prion diseases. • Disruption may explain prion protein mediated toxicity in Alzheimer's disease [ABSTRACT FROM AUTHOR]

Abstract: To identify molecular interaction partners of the cellular prion protein (PrPC), we applied a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the potassium channel tetramerization domain containing 1 (KCTD1) as a PrPC interacting protein. Deletion mapping showed that PrPC specifically binds KCTD1 through the unstructured PrP51-136 region. We further confirmed the interaction between PrPC and KCDT1 protein by co-immunoprecipitation in vivo and by a biosensor assay in vitro. Interestingly, the binding of an insertion mutant PrP8OR to KCTD1 is higher than that of wild-type PrPC, suggesting an important role for an unstructured region harboring octapeptide repeats in the KCTD1-PrPC interaction. Our results identify a novel PrPC-interacting protein and suggest a new approach to investigating the unidentified physiological cellular function of PrPC. [Copyright &y& Elsevier]

Abstract: Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrPC) is required for prion replication, the influence of PrPC abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrPC was overexpressed. Tg(MoPrP)4112 mice overexpressing PrPC supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrPC-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrPC is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrPSc amplification by using concentrated PrPC source and expands the use of this methodology. [Copyright &y& Elsevier]

Abstract: The prion protein is a cell surface glycoprotein whose physiological role remains elusive, while its implication in transmissible spongiform encephalopathies (TSEs) has been demonstrated. Multiple interactions between the prion protein and viruses have been described: viruses can act as co-factors in TSEs and life cycles of different viruses have been found to be controlled by prion modulation. We present data showing that human Adenovirus 5 induces prion expression. Inactivated Adenovirus did not alter prion transcription, while variants encoding for early products did, suggesting that the prion is stimulated by an early adenoviral function. Down-regulation of the prion through RNA interference showed that the prion controls adenovirus replication and expression. These data suggest that the prion protein could play a role in the defense strategy mounted by the host during viral infection, in a cell autonomous manner. These results have implications for the study of the prion protein and of associated TSEs. [Copyright &y& Elsevier]

Abstract: The cellular prion protein (PrPc) undergoes a physiological cleavage between amino acids 111 and 112, thereby leading to the secretion of an amino-terminal fragment referred to as N1. This proteolytic event is either constitutive or regulated by protein kinase C (PKC) and is operated by the disintegrins ADAM9/ADAM10 or ADAM17 respectively. We recently showed that the stimulation of the M1/M3 muscarinic receptors potentiates this cleavage via the phosphorylation and activation of ADAM17. We have examined the contribution of various PKC isoforms in the regulated processing of PrPc. First we show that the PDBu- and carbachol-stimulated N1 secretions are blocked by the general PKC inhibitor GF109203X. We establish that HEK293 and human-derived rhabdhomyosarcoma cells over-expressing constitutively active PKCα, PKCδ or PKCε, but not PKCζ, produce increased amounts of N1 and harbor enhanced ability to hydrolyze the fluorimetric substrate of ADAM17, JMV2770. Conversely, over-expression of the corresponding dominant negative proteins abolishes PDBU-stimulated N1 secretion and restores N1 to levels comparable to constitutive production. Moreover, deletion of PKCα lowers N1 recovery in primary cultured fibroblasts. Importantly, mutation of threonine 735 of ADAM17 significantly lowers the PDBu-induced N1 formation while transient over-expression of constitutively active PKCα, PKCδ or PKCε, but not PKCζ, induced both the phosphorylation of ADAM17 on its threonine residues and N1 secretion. As a corollary, T735A mutation concomitantly reversed PKCα-, PKCδ- and PKCε-induced ADAM17 phosphorylation and N1 recovery. Finally, we established that PKCε-dependent N1 production is fully prevented by ADAM17 deficiency. Altogether, the present results provide strong evidence that the activation of PKCα, δ and ε, but not ζ, isoforms leads to increased N1 secretion via the phosphorylation and activation of ADAM17, a process that likely accounts for M1/M3 muscarinic receptors-mediated control of N1 production. [Copyright &y& Elsevier]

Abstract: The cellular prion protein (PrPC) is highly conserved in the evolution of mammals, and therefore, thought to have important cellular functions. Despite decades of intensive research, the physiological function of PrPC remains enigmatic. We carried out a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the transmembrane protein tetraspanin-7 (CD231), as a PrPC interacting protein. We confirmed the interaction between PrPC and tetraspanin-7 by yeast two-hybrid assay, immunofluorescent co-localization, and immunocoprecipitation. Our mutational studies further demonstrated that PrPC specifically binds tetraspanin-7 through the region corresponding to bovine PrP154–182 containing alpha-helix 1. [Copyright &y& Elsevier]

Abstract: Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrPCWD of elk can readily induce the conversion of normal cervid PrP (PrPC) molecules to a protease-resistant form, but is less efficient in converting the PrPC of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrPCWD-induced conversion can be greatly enhanced in all species. Our results demonstrate that PrPC from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Moreover, partial denaturation of substrate PrPC can apparently overcome the structural barriers between more distant species. [Copyright &y& Elsevier]

Abstract: Cellular prion protein, PrPC, is essential for the development of prion diseases where it is considered to be a substrate for the formation of the disease-associated conformer, PrPSc. In sheep, PrPC is abundant in neuronal tissue and is also found at lower concentrations in a range of non-neuronal tissues, including mammary gland. Here, we demonstrate the presence of soluble PrPC in the non-cellular, non-lipid fraction of clarified ovine milk. Compared with brain-derived PrPC, ovine milk PrPC displays an increased electrophoretic mobility. Ovine milk PrPC is mainly present as three species that differ in the extent of their N-linked glycosylation, with glycoform profiles varying among animals. Similar PrPC species are also present in fresh and commercial homogenised/pasteurised bovine milk, with additional N-terminal PrPC fragments detectable in ruminant milk and commercial milk products. [Copyright &y& Elsevier]

Abstract: PrPC is a glycosylphosphatidylinositol (GPI) anchored glycoprotein of unknown function. Misfolding of normal cellular PrPC to the pathogenic PrPSc is the hallmark of prion diseases (transmissible spongiform encephalopathies). Prion diseases are characterized by extensive neurodegeneration and early death. Understanding how PrPC maintains its correct conformation is a major endeavor of current inquiry. Here we demonstrate a novel interaction between PrPC and the J protein family member, Rdj2 (DjA2; Dj3, Dnj3, Cpr3, and Hirip4). The importance of the J protein family in the cellular folding machinery has been recognized for many years. The PrPC/Rdj2 association was direct and concentration-dependent. Other J proteins such as CSPα and auxilin did not associate with PrPC in the absence of ATP, demonstrating the specificity of the PrPC/J protein interaction. These findings suggest that the J protein family serves as a ‘folding catalyst’ for PrPC and implicates Rdj2 as a factor in the protection against prion diseases. [Copyright &y& Elsevier]

The cellular prion protein (PrPc) is highly conserved in mammals and expressed widely in different tissues but its physiological role remains elusive. Recently, the human PrPc was shown to possess nucleic acid binding and chaperoning properties similar to human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, a key viral factor in virus structure and replication. These findings prompted us to determine if PrPc could influence HIV-1 replication. We used the human 293T cell line as a model system, since only a very low level of PrPc accumulates in these cells. Expression of PrP at a high level resulted in a specific decrease of HIV-1 Env and Vpr expression. Despite similar levels of intracellular Gag, virus production was reduced by eightfold and infectivity by three- to fourfold in the presence of PrPc. A PrPc mutant lacking the glycosylphosphatidylinositol (GPI) anchor peptide did not impair HIV-1 production, suggesting that PrPc trafficking is critical for this inhibitory effect. Coexpressing HIV-1 and PrPc in these cells also caused a fraction of PrPc to become partially proteinase K-resistant (PrPres), further illustrating the interactions between HIV-1 and PrPc. [Copyright &y& Elsevier]