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Liquid-liquid phase separation (LLPS) of the mammalian prion protein is mainly driven by its intrinsically disordered N-terminal domain (N-PrP). However, the specific intermolecular interactions that promote LLPS remain largely unknown. Here, we used extensive mutagenesis and comparative analyses of evolutionarily distant PrP species to gain insight into the relationship between protein sequence and phase behavior. LLPS of mouse PrP is dependent on two polybasic motifs in N-PrP that are conserved in all tetrapods. A unique feature of mammalian N-PrP is the octarepeat domain with four histidines that mediate binding to copper ions. We now show that the octarepeat is critical for promoting LLPS and preventing the formation of PrP aggregates. Amphibian N-PrP, which contains the polybasic motifs but lacks a repeat domain and histidines, does not undergo LLPS and forms nondynamic protein assemblies indicative of aggregates. Insertion of the mouse octarepeat domain restored LLPS of amphibian N-PrP, supporting its essential role in regulating the phase transition of PrP. This activity of the octarepeat domain was neither dependent on the four highly conserved histidines nor on copper binding. Instead, the regularly spaced tryptophan residues were critical for regulating LLPS, presumably via cation-π interactions with the polybasic motifs. Our study reveals a novel role for the tryptophan residues in the octarepeat in controlling phase transition of PrP and indicates that the ability of mammalian PrP to undergo LLPS has evolved with the octarepeat in the intrinsically disordered domain but independently of the histidines., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding., (© 2024 Banik et al.)

The mammalian prion protein (PrP C ) is composed of a large intrinsically disordered N-terminal and a structured C-terminal domain, containing three alpha-helical regions and a short, two-stranded beta-sheet. Traditionally, the activity of a protein was linked to the ability of the polypeptide chain to adopt a stable secondary/tertiary structure. This concept has been extended when it became evident that intrinsically disordered domains (IDDs) can participate in a broad range of defined physiological activities and play a major functional role in several protein classes including transcription factors, scaffold proteins, and signaling molecules. This ability of IDDs to engage in a variety of supramolecular complexes may explain the large number of PrP C -interacting proteins described. Here, we summarize diverse physiological and pathophysiological activities that have been described for the unstructured N-terminal domain of PrP C . In particular, we focus on subdomains that have been conserved in evolution.

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrP C ). PrP C is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrP C is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aβ oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aβ-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aβ-binding domain in this process., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Scrapie prion, PrP Sc , formation is the central event of all types of transmissible spongiform encephalopathies (TSEs), while the pathway with possible intermediates and their mechanism of formation from the normal isoform of prion (PrP), remains not fully understood. Recently, the G127V variant of the human PrP is reported to render the protein refractory to transmission of TSEs, via a yet unknown mechanism. Molecular dynamics studies suggested that this mutation interferes with the formation of PrP dimers. Here we analyze the dimerization of 127G and 127VPrP, in both in vitro and a mammalian cell culture system. Our results show that while molecular dynamics may capture the features affecting dimerization in vitro, G127V inhibiting dimer formation of PrP, these are not evidenced in a more complex cellular system.

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrP C ) into the scrapie isoform, denoted PrP Sc Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrP C and PrP Sc ; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrP C is converted in scrapie-infected cells, suggesting that not all PrP C species are suitable substrates for the conversion. On the basis of the observation that PrP C can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrP C , the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrP C Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrP C dimerization completely blocked its conversion into PrP Sc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrP C dimers had a dominant-negative inhibition effect on the conversion of monomeric PrP C Our findings suggest that PrP C monomers are the major substrates for PrP Sc propagation and that it may be possible to halt prion formation by stabilizing PrP C dimers., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Proteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer’s and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown., Methods: We produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches., Results: We show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell., Conclusions: With the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration.

The prion protein (PrP) is composed of two major domains of similar size. The structured C-terminal domain contains three alpha-helical regions and a short two-stranded beta-sheet, while the N-terminal domain is intrinsically disordered. The analysis of PrP mutants with deletions in the C-terminal globular domain provided the first hint that intrinsically disordered domains are inefficiently transported into the endoplasmic reticulum through the Sec61 translocon. Interestingly, C-terminally truncated PrP mutants have been linked to inherited prion disease in humans and are characterized by inefficient ER import and the formation of neurotoxic PrP conformers. In a recent study we found that the Sec61 translocon in eukaryotic cells as well as the SecY translocon in bacteria is inherently deficient in translocating intrinsically disordered proteins. Moreover, our results suggest that translocon-associated components in eukaryotic cells enable the Sec61 complex to transport secretory proteins with extended unstructured domains such as PrP and shadoo.

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

PrP C , the cellular isoform of the prion protein, serves to transduce the neurotoxic effects of PrP Sc , the infectious isoform, but how this occurs is mysterious. Here, using a combination of electrophysiological, cellular, and biophysical techniques, we show that the flexible, N-terminal domain of PrP C functions as a powerful toxicity-transducing effector whose activity is tightly regulated in cis by the globular C-terminal domain. Ligands binding to the N-terminal domain abolish the spontaneous ionic currents associated with neurotoxic mutants of PrP, and the isolated N-terminal domain induces currents when expressed in the absence of the C-terminal domain. Anti-PrP antibodies targeting epitopes in the C-terminal domain induce currents, and cause degeneration of dendrites on murine hippocampal neurons, effects that entirely dependent on the effector function of the N-terminus. NMR experiments demonstrate intramolecular docking between N- and C-terminal domains of PrP C , revealing a novel auto-inhibitory mechanism that regulates the functional activity of PrP C .

Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrP(C)) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrP(Sc) leading to prion diseases. We show that a deletion in the C-terminal domain of PrP(C) (PrPΔ214-229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrP(C). Transgenic (Tg(PrPΔ214-229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214-229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice.