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.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

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.

NEMO reshapes the α-Synuclein aggregate interface and acts as an autophagy adapter by co-condensation with p62.

NEMO is a ubiquitin-binding protein which regulates canonical NF-κB pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identify an NF-κB-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO-encoding IKBKG gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including α-synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at α-synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62.

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.

Linear ubiquitination induces NEMO phase separation to activate NF-κB signaling.

The NF-κB essential modulator NEMO is the core regulatory component of the inhibitor of κB kinase complex, which is a critical checkpoint in canonical NF-κB signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-1ß, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and the formation of NEMO assemblies in cells after exposure to IL-1ß. Phase separation is promoted by both binding of NEMO to linear ubiquitin chains and covalent linkage of M1-ubiquitin to NEMO and is essential but not sufficient for its phase separation. Supporting the functional relevance of NEMO phase separation in signaling, a pathogenic NEMO mutant, which is impaired in both binding and linkage to linear ubiquitin chains, does not undergo phase separation and is defective in mediating IL-1ß-induced NF-κB activation.

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

Bivalent metal ions induce formation of α-synuclein fibril polymorphs with different cytotoxicities.

α-Synuclein (α-Syn) aggregates are key components of intracellular inclusion bodies characteristic of Parkinson's disease (PD) and other synucleinopathies. Metal ions have been considered as the important etiological factors in PD since their interactions with α-Syn alter the kinetics of fibrillation. In the present study, we have systematically explored the effects of Zn2+, Cu2+, Ca2+, and Mg2+ cations on α-Syn fibril formation. Specifically, we determined fibrillation kinetics, size, morphology, and secondary structure of the fibrils and their cytotoxic activity. While all cations accelerate fibrillation, we observed distinct effects of the different ions. For example, Zn2+ induced fibrillation by lower tlag and higher kapp and formation of shorter fibrils, while Ca2+ ions lead to formation of longer fibrils, as evidenced by dynamic light scattering and atomic force microscopy studies. Additionally, the morphology of formed fibrils was different. Circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopies revealed higher contents of ß-sheets in fibrils. Interestingly, cell viability studies indicated nontoxicity of α-Syn fibrils formed in the presence of Zn2+ ions, while the fibrils formed in the presence of Cu2+, Ca2+, and Mg2+ were cytotoxic. Our results revealed that α-Syn fibrils formed in the presence of different divalent cations have distinct structural and cytotoxic features.

Ligands binding to the prion protein induce its proteolytic release with therapeutic potential in neurodegenerative proteinopathies.

The prion protein (PrPC) is a central player in neurodegenerative diseases, such as prion diseases or Alzheimer's disease. In contrast to disease-promoting cell surface PrPC, extracellular fragments act neuroprotective by blocking neurotoxic disease-associated protein conformers. Fittingly, PrPC release by the metalloprotease ADAM10 represents a protective mechanism. We used biochemical, cell biological, morphological, and structural methods to investigate mechanisms stimulating this proteolytic shedding. Shed PrP negatively correlates with prion conversion and is markedly redistributed in murine brain in the presence of prion deposits or amyloid plaques, indicating a sequestrating activity. PrP-directed ligands cause structural changes in PrPC and increased shedding in cells and organotypic brain slice cultures. As an exception, some PrP-directed antibodies targeting repetitive epitopes do not cause shedding but surface clustering, endocytosis, and degradation of PrPC. Both mechanisms may contribute to beneficial actions described for PrP-directed ligands and pave the way for new therapeutic strategies against currently incurable neurodegenerative diseases.

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.

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III.

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.

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.

The G127V variant of the prion protein interferes with dimer formation in vitro but not in cellulo.

Scrapie prion, PrPSc, 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.

The key role of solvent in condensation: Mapping water in liquid-liquid phase-separated FUS.

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has emerged as a pervasive principle in cell biology, allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that form condensates under physiological conditions often contain intrinsically disordered regions with low-complexity domains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dilute aqueous solution. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liquid-liquid phase-separated droplets to provide experimental evidence for the key role of the solvent as a thermodynamic driving force. The FUS concentration inside LLPS droplets was determined to be increased to 2.0 mM independent of the initial protein concentration (5 or 10 µM solutions) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-separated FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in hand with enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodynamic driving force in phase separation.

SecY-mediated quality control prevents the translocation of non-gated porins.

OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm.

Transgenic Overexpression of the Disordered Prion Protein N1 Fragment in Mice Does Not Protect Against Neurodegenerative Diseases Due to Impaired ER Translocation.

The structurally disordered N-terminal half of the prion protein (PrPC) is constitutively released into the extracellular space by an endogenous proteolytic cleavage event. Once liberated, this N1 fragment acts neuroprotective in ischemic conditions and interferes with toxic peptides associated with neurodegenerative diseases, such as amyloid-beta (Aß) in Alzheimer's disease. Since analog protective effects of N1 in prion diseases, such as Creutzfeldt-Jakob disease, have not been studied, and given that the protease releasing N1 has not been identified to date, we have generated and characterized transgenic mice overexpressing N1 (TgN1). Upon intracerebral inoculation of TgN1 mice with prions, no protective effects were observed at the levels of survival, clinical course, neuropathological, or molecular assessment. Likewise, primary neurons of these mice did not show protection against Aß toxicity. Our biochemical and morphological analyses revealed that this lack of protective effects is seemingly due to an impaired ER translocation of the disordered N1 resulting in its cytosolic retention with an uncleaved signal peptide. Thus, TgN1 mice represent the first animal model to prove the inefficient ER translocation of intrinsically disordered domains (IDD). In contrast to earlier studies, our data challenge roles of cytoplasmic N1 as a cell penetrating peptide or as a potent "anti-prion" agent. Lastly, our study highlights both the importance of structured domains in the nascent chain for proteins to be translocated and aspects to be considered when devising novel N1-based therapeutic approaches against neurodegenerative diseases.

The parkin-coregulated gene product PACRG promotes TNF signaling by stabilizing LUBAC.

The Parkin-coregulated gene (PACRG), which encodes a protein of unknown function, shares a bidirectional promoter with Parkin (PRKN), which encodes an E3 ubiquitin ligase. Because PRKN is important in mitochondrial quality control and protection against stress, we tested whether PACRG also affected these pathways in various cultured human cell lines and in mouse embryonic fibroblasts. PACRG did not play a role in mitophagy but did play a role in tumor necrosis factor (TNF) signaling. Similarly to Parkin, PACRG promoted nuclear factor κB (NF-κB) activation in response to TNF. TNF-induced nuclear translocation of the NF-κB subunit p65 and NF-κB-dependent transcription were decreased in PACRG-deficient cells. Defective canonical NF-κB activation in the absence of PACRG was accompanied by a decrease in linear ubiquitylation mediated by the linear ubiquitin chain assembly complex (LUBAC), which is composed of the two E3 ubiquitin ligases HOIP and HOIL-1L and the adaptor protein SHARPIN. Upon TNF stimulation, PACRG was recruited to the activated TNF receptor complex and interacted with LUBAC components. PACRG functionally replaced SHARPIN in this context. In SHARPIN-deficient cells, PACRG prevented LUBAC destabilization, restored HOIP-dependent linear ubiquitylation, and protected cells from TNF-induced apoptosis. This function of PACRG in positively regulating TNF signaling may help to explain the association of PACRG and PRKN polymorphisms with an increased susceptibility to intracellular pathogens.

A protein quality control pathway regulated by linear ubiquitination.

Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unwanted interactions, for example with the low complexity sequence domain-containing transcription factor Sp1, and proteasomal degradation of misfolded Huntingtin is facilitated. Notably, all three core LUBAC components are transcriptionally regulated by Sp1, linking defective LUBAC expression to Huntington's disease. In support of a protective activity of linear ubiquitination, silencing of OTULIN, a deubiquitinase with unique specificity for linear polyubiquitin, decreases proteotoxicity, whereas silencing of HOIP has the opposite effect. These findings identify linear ubiquitination as a protein quality control mechanism and hence a novel target for disease-modifying strategies in proteinopathies.

The signal peptide plus a cluster of positive charges in prion protein dictate chaperone-mediated Sec61 channel gating.

The Sec61-complex as a dynamic polypeptide-conducting channel mediates protein transport into the human endoplasmic reticulum (ER) with the help of additional components. ER membrane resident Hsp40-type co-chaperone Sec63 as well as the ER lumenal Hsp70-type chaperone BiP were proposed to facilitate channel opening in a precursor-specific fashion. Here, we report on their rules of engagement in ER import of the prion protein (PrP) by addressing sixteen PrP-related variants which differ in their signal peptides and mature parts, respectively. Transport into the ER of semi-permeabilized human cells was analyzed upon depletion of the components by siRNA- or toxin-treatment. The results are consistent with the view of separate functions of BiP and Sec63 and strongly suggest that the co-chaperone/chaperone-pair facilitates Sec61 channel gating to the open state when precursor polypeptides with weak signal peptides in combination with detrimental features in the adjacent mature part were targeted. Thus, we expand the view of chaperone-mediated Sec61 channel gating by providing a novel example of a polybasic motif that interferes with signal peptide-mediated Sec61 channel gating. This article has an associated First Person interview with the first author of the paper.