A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation.

Many organisms evolved strategies to survive desiccation. Plant seeds protect dehydrated embryos from various stressors and can lay dormant for millennia. Hydration is the key trigger to initiate germination, but the mechanism by which seeds sense water remains unresolved. We identified an uncharacterized Arabidopsis thaliana prion-like protein we named FLOE1, which phase separates upon hydration and allows the embryo to sense water stress. We demonstrate that biophysical states of FLOE1 condensates modulate its biological function in vivo in suppressing seed germination under unfavorable environments. We find intragenic, intraspecific, and interspecific natural variation in FLOE1 expression and phase separation and show that intragenic variation is associated with adaptive germination strategies in natural populations. This combination of molecular, organismal, and ecological studies uncovers FLOE1 as a tunable environmental sensor with direct implications for the design of drought-resistant crops, in the face of climate change.

Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer's Disease.

To elucidate the role of Tau isoforms and post-translational modification (PTM) stoichiometry in Alzheimer's disease (AD), we generated a high-resolution quantitative proteomics map of 95 PTMs on multiple isoforms of Tau isolated from postmortem human tissue from 49 AD and 42 control subjects. Although Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and frequency for AD, suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner, leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by analysis of size-fractionated Tau. To summarize, features in the Tau protein critical for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C terminus, an increase in negative charge in the proline-rich region (PRR), and a decrease in positive charge in the microtubule binding domain (MBD).

Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-ß/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.

A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins.

Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Precision proteoform design for 4R tau isoform selective templated aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

Functional Domains of NEAT1 Architectural lncRNA Induce Paraspeckle Assembly through Phase Separation.

A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.

An interchangeable prion-like domain is required for Ty1 retrotransposition.

Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. Saccharomyces cerevisiae and its close relatives harbor several families of LTR-retrotransposons, the most abundant being Ty1 in several laboratory strains. The cytosolic foci that nucleate Ty1 virus-like particle (VLP) assembly are not well understood. These foci, termed retrosomes or T-bodies, contain Ty1 Gag and likely Gag-Pol and the Ty1 mRNA destined for reverse transcription. Here, we report an intrinsically disordered N-terminal prion-like domain (PrLD) within Gag that is required for transposition. This domain contains amino acid composition similar to known yeast prions and is sufficient to nucleate prionogenesis in an established cell-based prion reporter system. Deleting the Ty1 PrLD results in dramatic VLP assembly and retrotransposition defects but does not affect Gag protein level. Ty1 Gag chimeras in which the PrLD is replaced with other sequences, including yeast and mammalian prionogenic domains, display a range of retrotransposition phenotypes from wild type to null. We examine these chimeras throughout the Ty1 replication cycle and find that some support retrosome formation, VLP assembly, and retrotransposition, including the yeast Sup35 prion and the mouse PrP prion. Our interchangeable Ty1 system provides a useful, genetically tractable in vivo platform for studying PrLDs, complete with a suite of robust and sensitive assays. Our work also invites study into the prevalence of PrLDs in additional mobile elements.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

FUS and TDP-43 Phases in Health and Disease.

The distinct prion-like domains (PrLDs) of FUS and TDP-43, modulate phase transitions that result in condensates with a range of material states. These assemblies are implicated in both health and disease. In this review, we examine how sequence, structure, post-translational modifications, and RNA can affect the self-assembly of these RNA-binding proteins (RBPs). We discuss how our emerging understanding of FUS and TDP-43 liquid-liquid phase separation (LLPS) and aggregation, could be leveraged to design new therapies for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and limbic-predominant age-related TDP-43 encephalopathy (LATE).

Impairment of the Glial Phagolysosomal System Drives Prion-Like Propagation in a Drosophila Model of Huntington's Disease.

Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative disease pathogenesis. Phagocytic glia are responsible for regulating the load of pathological proteins in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. A forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings uncover new mechanisms that enhance our understanding of the beneficial and harmful effects of phagocytic glia in HD and other neurodegenerative diseases.

Alzheimer's disease seeded tau forms paired helical filaments yet lacks seeding potential.

Alzheimer's disease (AD) and many other neurodegenerative diseases are characterized by pathological aggregation of the protein tau. These tau aggregates spread in a stereotypical spatiotemporal pattern in the brain of each disease, suggesting that the misfolded tau can recruit soluble monomers to adopt the same pathological structure. To investigate whether recruited tau indeed adopts the same structure and properties as the original seed, here we template recombinant full-length 0N3R tau, 0N4R tau, and an equimolar mixture of the two using sarkosyl-insoluble tau extracted from AD brain and determine the structures of the resulting fibrils using cryoelectron microscopy. We show that these cell-free amplified tau fibrils adopt the same molecular structure as the AD paired-helical filament (PHF) tau but are unable to template additional monomers. Therefore, the PHF structure alone is insufficient for defining the pathological properties of AD tau, and other biochemical components such as tau posttranslational modifications, other proteins, polyanionic cofactors, and salt are required for the prion-like serial propagation of tauopathies.

Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1G85R-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease.

Tracing the Evolutionary History of the Temperature-Sensing Prion-like Domain in EARLY FLOWERING 3 Highlights the Uniqueness of AtELF3.

Plants have evolved mechanisms to anticipate and adjust their growth and development in response to environmental changes. Understanding the key regulators of plant performance is crucial to mitigate the negative influence of global climate change on crop production. EARLY FLOWERING 3 (ELF3) is one such regulator playing a critical role in the circadian clock and thermomorphogenesis. In Arabidopsis thaliana, ELF3 contains a prion-like domain (PrLD) that acts as a thermosensor, facilitating liquid-liquid phase separation at high ambient temperatures. To assess the conservation of this function across the plant kingdom, we traced the evolutionary emergence of ELF3, with a focus on the presence of PrLDs. We found that the PrLD, primarily influenced by the length of polyglutamine (polyQ) repeats, is most prominent in Brassicales. Analyzing 319 natural A. thaliana accessions, we confirmed the previously described wide range of polyQ length variation in AtELF3, but found it to be only weakly associated with geographic origin, climate conditions, and classic temperature-responsive phenotypes. Interestingly, similar polyQ length variation was not observed in several other investigated Bassicaceae species. Based on these findings, available prediction tools and limited experimental evidence, we conclude that the emergence of PrLD, and particularly polyQ length variation, is unlikely to be a key driver of environmental adaptation. Instead, it likely adds an additional layer to ELF3's role in thermomorphogenesis in A. thaliana, with its relevance in other species yet to be confirmed.

Propagation of Tau Aggregates and Neurodegeneration.

A pathway from the natively unfolded microtubule-associated protein Tau to a highly structured amyloid fibril underlies human Tauopathies. This ordered assembly causes disease and represents the gain of toxic function. In recent years, evidence has accumulated to suggest that Tau inclusions form first in a small number of brain cells, from where they propagate to other regions, resulting in neurodegeneration and disease. Propagation of pathology is often called prion-like, which refers to the capacity of an assembled protein to induce the same abnormal conformation in a protein of the same kind, initiating a self-amplifying cascade. In addition, prion-like encompasses the release of protein aggregates from brain cells and their uptake by neighboring cells. In mice, the intracerebral injection of Tau inclusions induces the ordered assembly of monomeric Tau, followed by its spreading to distant brain regions. Conformational differences between Tau aggregates from transgenic mouse brain and in vitro assembled recombinant protein account for the greater seeding potency of brain aggregates. Short fibrils constitute the major species of seed-competent Tau in the brains of transgenic mice. The existence of multiple human Tauopathies with distinct fibril morphologies has led to the suggestion that different molecular conformers (or strains) of aggregated Tau exist.

Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics.

Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

Structural Variations of Prions and Prion-like Proteins Associated with Neurodegeneration.

Neurodegeneration is becoming one of the leading causes of death worldwide as the population expands and grows older. There is a growing desire to understand the mechanisms behind prion proteins as well as the prion-like proteins that make up neurodegenerative diseases (NDs), including Alzheimer's disease (AD) and Parkinson's disease (PD). Both amyloid-ß (Aß) and hyperphosphorylated tau (p-tau) proteins behave in ways similar to those of the infectious form of the prion protein, PrPSc, such as aggregating, seeding, and replicating under not yet fully understood mechanisms, thus the designation of prion-like. This review aims to highlight the shared mechanisms between prion-like proteins and prion proteins in the structural variations associated with aggregation and disease development. These mechanisms largely focus on the dysregulation of protein homeostasis, self-replication, and protein aggregation, and this knowledge could contribute to diagnoses and treatments for the given NDs.

The HSP110/HSP70 disaggregation system generates spreading-competent toxic α-synuclein species.

The accumulation and prion-like propagation of α-synuclein and other amyloidogenic proteins are associated with devastating neurodegenerative diseases. Metazoan heat shock protein HSP70 and its co-chaperones DNAJB1 and HSP110 constitute a disaggregation machinery that is able to disassemble α-synuclein fibrils in vitro, but its physiological effects on α-synuclein toxicity are unknown. Here, we depleted Caenorhabditis elegans HSP-110 and monitored the consequences on α-synuclein-related pathological phenotypes such as misfolding, intercellular spreading, and toxicity in C. elegans in vivo models. Depletion of HSP-110 impaired HSP70 disaggregation activity, prevented resolubilization of amorphous aggregates, and compromised the overall cellular folding capacity. At the same time, HSP-110 depletion reduced α-synuclein foci formation, cell-to-cell transmission, and toxicity. These data demonstrate that the HSP70 disaggregation activity constitutes a double-edged sword, as it is essential for maintaining cellular proteostasis but also involved in the generation of toxic amyloid-type protein species.

In silico analysis of fungal prion-like proteins for elucidating their role in plant-fungi interactions.

Prion-like proteins (PrLPs) have emerged as beneficial molecules with implications in adaptive responses. These proteins possess a conserved prion-like domain (PrLD) which is an intrinsically disordered region capable of adopting different conformations upon perceiving external stimuli. Owing to changes in protein conformation, functional characteristics of proteins harboring PrLDs get altered thereby, providing a unique mode of protein-based regulation. Since PrLPs are ubiquitous in nature and involved in diverse functions, through this study, we aim to explore the role of such domains in yet another important physiological process viz. plant-microbe interactions to get insights into the mechanisms dictating cross-kingdom interactions. We have evaluated the presence and functions of PrLPs in 18 different plant-associated fungi of agricultural importance to unravel their role in plant-microbe interactions. Of the 241,997 proteins scanned, 3,820 (~ 1.6%) were identified as putative PrLPs with pathogenic fungi showing significantly higher PrLP density than their beneficial counterparts. Further, through GO enrichment analysis, we could predict several PrLPs from pathogenic fungi to be involved in virulence and formation of stress granules. Notably, PrLPs involved in (retro)transposition were observed exclusively in pathogenic fungi. We even analyzed publicly available data for the expression alterations of fungal PrLPs upon their interaction with their respective hosts which revealed perturbation in the levels of some PrLP-encoding genes during interactions with plants. Overall, our work sheds light into the probable role of prion-like candidates in plant-fungi interaction, particularly in context of pathogenesis, paving way for more focused studies for validating their role.

Isotope-labeled amyloid-ß does not transmit to the brain in a prion-like manner after peripheral administration.

Findings of early cerebral amyloid-ß deposition in mice after peripheral injection of amyloid-ß-containing brain extracts, and in humans following cadaveric human growth hormone treatment raised concerns that amyloid-ß aggregates and possibly Alzheimer's disease may be transmissible between individuals. Yet, proof that Aß actually reaches the brain from the peripheral injection site is lacking. Here, we use a proteomic approach combining stable isotope labeling of mammals and targeted mass spectrometry. Specifically, we generate 13 C-isotope-labeled brain extracts from mice expressing human amyloid-ß and track 13 C-lysine-labeled amyloid-ß after intraperitoneal administration into young amyloid precursor protein-transgenic mice. We detect injected amyloid-ß in the liver and lymphoid tissues for up to 100 days. In contrast, injected 13 C-lysine-labeled amyloid-ß is not detectable in the brain whereas the mice incorporate 13 C-lysine from the donor brain extracts into endogenous amyloid-ß. Using a highly sensitive and specific proteomic approach, we demonstrate that amyloid-ß does not reach the brain from the periphery. Our study argues against potential transmissibility of Alzheimer's disease while opening new avenues to uncover mechanisms of pathophysiological protein deposition.