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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., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

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., (© 2019 The Authors.)

Protein targeting to specified cellular compartments is essential to maintain cell function and homeostasis. In eukaryotic cells, two major pathways rely on N-terminal signal peptides to target proteins to either the endoplasmic reticulum (ER) or mitochondria. In this study, we show that the ER signal peptides of the prion protein-like protein shadoo, the neuropeptide hormone somatostatin and the amyloid precursor protein have the property to mediate alternative targeting to mitochondria. Remarkably, the targeting direction of these signal peptides is determined by structural elements within the nascent chain. Each of the identified signal peptides promotes efficient ER import of nascent chains containing α-helical domains, but targets unstructured polypeptides to mitochondria. Moreover, we observed that mitochondrial targeting by the ER signal peptides correlates inversely with ER import efficiency. When ER import is compromised, targeting to mitochondria is enhanced, whereas improving ER import efficiency decreases mitochondrial targeting. In conclusion, our study reveals a novel mechanism of dual targeting to either the ER or mitochondria that is mediated by structural features within the nascent chain.

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a β-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various β-sheet-rich (β) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid β-peptide, (iii) yeast prion proteins or (iv) designed β-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with β-conformers. Interestingly, a secreted version of N-PrP associated with β-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various β-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.

Studies in transgenic mice revealed that neurodegeneration induced by scrapie prion (PrP(Sc)) propagation is dependent on neuronal expression of the cellular prion protein PrP(C). On the other hand, there is evidence that PrP(C) itself has a stress-protective activity. Here, we show that the toxic activity of PrP(Sc) and the protective activity of PrP(C) are interconnected. With a novel co-cultivation assay, we demonstrate that PrP(Sc) can induce apoptotic signalling in PrP(C)-expressing cells. However, cells expressing PrP mutants with an impaired stress-protective activity were resistant to PrP(Sc)-induced toxicity. We also show that the internal hydrophobic domain promotes dimer formation of PrP and that dimerization of PrP is linked to its stress-protective activity. PrP mutants defective in dimer formation did not confer enhanced stress tolerance. Moreover, in chronically scrapie-infected neuroblastoma cells the amount of PrP(C) dimers inversely correlated with the amount of PrP(Sc) and the resistance of the cells to various stress conditions. Our results provide new insight into the mechanism of PrP(C)-mediated neuroprotection and indicate that pathological PrP conformers abuse PrP(C)-dependent pathways for apoptotic signalling.

The etiologies of neurodegenerative diseases may be diverse; however, a common pathological denominator is the formation of aberrant protein conformers and the occurrence of pathognomonic proteinaceous deposits. Different approaches coming from neuropathology, genetics, animal modeling and biophysics have established a crucial role of protein misfolding in the pathogenic process. However, there is an ongoing debate about the nature of the harmful proteinaceous species and how toxic conformers selectively damage neuronal populations. Increasing evidence indicates that soluble oligomers are associated with early pathological alterations, and strikingly, oligomeric assemblies of different disease-associated proteins may share common structural features. A major step towards the understanding of mechanisms implicated in neuronal degeneration is the identification of genes, which are responsible for familial variants of neurodegenerative diseases. Studies based on these disease-associated genes illuminated the two faces of protein misfolding in neurodegeneration: a gain of toxic function and a loss of physiological function, which can even occur in combination. Here, we summarize how these two faces of protein misfolding contribute to the pathomechanisms of Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease and prion diseases.

Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We show that the chemical compound Suramin induced aggregation of PrP in a post-ER/Golgi compartment and prevented further trafficking of PrP(c) to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re-routed to acidic compartments for intracellular degradation. In contrast to PrP(Sc) in prion-infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin-induced intracellular re-routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti-prion compounds.

The fundamental event in prion diseases involves a conformational change in one or more of the alpha-helices of the cellular prion protein (PrP(C)) as they are converted into beta-sheets during the formation of the pathogenic isoform (PrP(Sc)). Here, we show that exposure of scrapie-infected mouse neuroblastoma (ScN2a) cells to reagents known to stabilize proteins in their native conformation reduced the rate and extent of PrP(Sc) formation. Such reagents include the cellular osmolytes glycerol and trimethylamine N-oxide (TMAO) and the organic solvent dimethylsulfoxide (DMSO), which we refer to as 'chemical chaperones' because of their influence on protein folding. Although the chemical chaperones did not appear to affect the existing population of PrP(Sc) molecules in ScN2a cells, they did interfere with the formation of PrP(Sc) from newly synthesized PrP(C). We suggest that the chemical chaperones act to stabilize the alpha-helical conformation of PrP(C) and thereby prevent the protein from undergoing a conformational change to produce PrP(Sc). These observations provide further support for the idea that prions arise due to a change in protein conformation and reveal potential strategies for preventing PrP(Sc) formation.