VCP/p97 mediates nuclear targeting of non-ER-imported prion protein to maintain proteostasis.

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.

Liquid-liquid phase separation of the prion protein is regulated by the octarepeat domain independently of histidines and copper.

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 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 non-dynamic protein assemblies indicative of aggregates. Insertion of the mouse octarepeat domain restored LLPS of amphibian N-PrP, supporting its essential role in regulating 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.

Cross-seeding by prion protein inactivates TDP-43.

A common pathological denominator of various neurodegenerative diseases is the accumulation of protein aggregates. Neurotoxic effects are caused by a loss of the physiological activity of the aggregating protein and/or a gain of toxic function of the misfolded protein conformers. In transmissible spongiform encephalopathies or prion diseases, neurodegeneration is caused by aberrantly folded isoforms of the prion protein (PrP). However, it is poorly understood how pathogenic PrP conformers interfere with neuronal viability. Employing in vitro approaches, cell culture, animal models and patients' brain samples, we show that misfolded PrP can induce aggregation and inactivation of TAR DNA-binding protein-43 (TDP-43). Purified PrP aggregates interact with TDP-43 in vitro and in cells and induce the conversion of soluble TDP-43 into non-dynamic protein assemblies. Similarly, mislocalized PrP conformers in the cytosol bind to and sequester TDP-43 in cytosolic aggregates. As a consequence, TDP-43-dependent splicing activity in the nucleus is significantly decreased, leading to altered protein expression in cells with cytosolic PrP aggregates. Finally, we present evidence for cytosolic TDP-43 aggregates in neurons of transgenic flies expressing mammalian PrP and Creutzfeldt-Jakob disease patients. Our study identified a novel mechanism of how aberrant PrP conformers impair physiological pathways by cross-seeding.

Hydration makes a difference! How to tune protein complexes between liquid-liquid and liquid-solid phase separation.

Understanding how protein rich condensates formed upon liquid-liquid phase separation (LLPS) evolve into solid aggregates is of fundamental importance for several medical applications, since these are suspected to be hot-spots for many neurotoxic diseases. This requires developing experimental approaches to observe in real-time both LLPS and liquid-solid phase separation (LSPS), and to unravel the delicate balance of protein and water interactions dictating the free energy differences between the two. We present a vibrational THz spectroscopy approach that allows doing so from the point of view of hydration water. We focus on a cellular prion protein of high medical relevance, which we can drive to undergo either LLPS or LSPS with few mutations. We find that it is a subtle balance of hydrophobic and hydrophilic solvation contributions that allows tuning between LLPS and LSPS. Hydrophobic hydration provides an entropic driving force to phase separation, through the release of hydration water into the bulk. Water hydrating hydrophilic groups provides an enthalpic driving force to keep the condensates in a liquid state. As a result, when we modify the protein by a few mutations to be less hydrophilic, we shift from LLPS to LSPS. This molecular understanding paves the way for a rational design of proteins.

Biological Functions of the Intrinsically Disordered N-Terminal Domain of the Prion Protein: A Possible Role of Liquid-Liquid Phase Separation.

The mammalian prion protein (PrPC) 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 PrPC-interacting proteins described. Here, we summarize diverse physiological and pathophysiological activities that have been described for the unstructured N-terminal domain of PrPC. In particular, we focus on subdomains that have been conserved in evolution.

The N-terminal domain of the prion protein is required and sufficient for liquid-liquid phase separation: A crucial role of the Aß-binding domain.

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 (PrPC). PrPC is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrPC 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 Aß 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 Aß-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 Aß-binding domain in this process.