Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains.

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

You come at the misfolded proteins, you best not miss.

A recent study by Huang et al. unexpectedly uncovered that DAXX moonlights as a booster of protein folding, including counteracting aggregation of tumor suppressor p53. Since p53 aggregation is a common hallmark of cancer, this finding provides a potential pathway to therapeutically reactivate p53 signaling and halt tumor progression.

Cellular Handling of Protein Aggregates by Disaggregation Machines.

Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins.

The Saccharomyces cerevisiae Yta7 ATPase hexamer contains a unique bromodomain tier that functions in nucleosome disassembly.

The Saccharomyces cerevisiae Yta7 is a chromatin remodeler harboring a histone-interacting bromodomain (BRD) and two AAA+ modules. It is not well understood how Yta7 recognizes the histone H3 tail to promote nucleosome disassembly for DNA replication or RNA transcription. By cryo-EM analysis, here we show that Yta7 assembles a three-tiered hexamer with a top BRD tier, a middle AAA1 tier, and a bottom AAA2 tier. Unexpectedly, the Yta7 BRD stabilizes a four-stranded ß-helix, termed BRD-interacting motif (BIM), of the largely disordered N-terminal region. The BIM motif is unique to the baker's yeast, and we show both BRD and BIM contribute to nucleosome recognition. We found that Yta7 binds both acetylated and nonacetylated H3 peptides but with a higher affinity for the unmodified peptide. This property is consistent with the absence of key residues of canonical BRDs involved in acetylated peptide recognition and the role of Yta7 in general nucleosome remodeling. Interestingly, the BRD tier exists in a spiral and a flat-ring form on top of the Yta7 AAA+ hexamer. The spiral is likely in a nucleosome-searching mode because the bottom BRD blocks the entry to the AAA+ chamber. The flat ring may be in a nucleosome disassembly state because the entry is unblocked and the H3 peptide has entered the AAA+ chamber and is stabilized by the AAA1 pore loops 1 and 2. Indeed, we show that the BRD tier is a flat ring when bound to the nucleosome. Overall, our study sheds light on the nucleosome disassembly by Yta7.

A minimal construct of nuclear-import receptor Karyopherin-ß2 defines the regions critical for chaperone and disaggregation activity.

Karyopherin-ß2 (Kapß2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapß2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Remarkably, Kapß2 prevents and reverses aberrant phase transitions of these cargoes, which is cytoprotective. However, the molecular determinants of Kapß2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapß2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapß2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8 to 20 of Kapß2 recapitulate all salient features of Kapß2 activity. By contrast, Kapß2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapß2 construct for delivery in adeno-associated viruses as a potential therapeutic for amyotrophic lateral sclerosis/frontotemporal dementia, multisystem proteinopathy, and related disorders.

Nucleotide Exchange Factors for Hsp70 Molecular Chaperones: GrpE, Hsp110/Grp170, HspBP1/Sil1, and BAG Domain Proteins.

Molecular chaperones of the Hsp70 family are key components of the cellular protein-folding machinery. Substrate folding is accomplished by iterative cycles of ATP binding, hydrolysis, and release. The ATPase activity of Hsp70 is regulated by two main classes of cochaperones: J-domain proteins stimulate ATPase hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) facilitate the conversion from the ADP-bound to the ATP-bound state, thus closing the chaperone folding cycle. NEF function can additionally be antagonized by ADP dissociation inhibitors. Beginning with the discovery of the prototypical bacterial NEF, GrpE, a large diversity of nucleotide exchange factors for Hsp70 have been identified, connecting it to a multitude of cellular processes in the eukaryotic cell. Here we review recent advances toward structure and function of nucleotide exchange factors from the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families and discuss how these cochaperones connect protein folding with cellular quality control and degradation pathways.

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species.

α-synuclein aggregation is present in Parkinson's disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid.

The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K-12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K-12, the 4.88 Mbp Fec10 genome is characterized by distinct single-nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two-component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpGGI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand-alone disaggregation activity, but observed distinct biochemical characteristics of ClpGGI-Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpGGI-SG17M. Furthermore, we noted a unique contribution of ClpGGI-Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food-borne heat-resistant strains in the human gut.

Deciphering the mechanism and function of Hsp100 unfoldases from protein structure.

Hsp100 chaperones, also known as Clp proteins, constitute a family of ring-forming ATPases that differ in 3D structure and cellular function from other stress-inducible molecular chaperones. While the vast majority of ATP-dependent molecular chaperones promote the folding of either the nascent chain or a newly imported polypeptide to reach its native conformation, Hsp100 chaperones harness metabolic energy to perform the reverse and facilitate the unfolding of a misfolded polypeptide or protein aggregate. It is now known that inside cells and organelles, different Hsp100 members are involved in rescuing stress-damaged proteins from a previously aggregated state or in recycling polypeptides marked for degradation. Protein degradation is mediated by a barrel-shaped peptidase that physically associates with the Hsp100 hexamer to form a two-component system. Notable examples include the ClpA:ClpP (ClpAP) and ClpX:ClpP (ClpXP) proteases that resemble the ring-forming FtsH and Lon proteases, which unlike ClpAP and ClpXP, feature the ATP-binding and proteolytic domains in a single polypeptide chain. Recent advances in electron cryomicroscopy (cryoEM) together with single-molecule biophysical studies have now provided new mechanistic insight into the structure and function of this remarkable group of macromolecular machines.

Hsp110 mitigates α-synuclein pathology in vivo.

Parkinson's disease is characterized by the aggregation of the presynaptic protein α-synuclein and its deposition into pathologic Lewy bodies. While extensive research has been carried out on mediators of α-synuclein aggregation, molecular facilitators of α-synuclein disaggregation are still generally unknown. We investigated the role of molecular chaperones in both preventing and disaggregating α-synuclein oligomers and fibrils, with a focus on the mammalian disaggregase complex. Here, we show that overexpression of the chaperone Hsp110 is sufficient to reduce α-synuclein aggregation in a mammalian cell culture model. Additionally, we demonstrate that Hsp110 effectively mitigates α-synuclein pathology in vivo through the characterization of transgenic Hsp110 and double-transgenic α-synuclein/Hsp110 mouse models. Unbiased analysis of the synaptic proteome of these mice revealed that overexpression of Hsp110 can override the protein changes driven by the α-synuclein transgene. Furthermore, overexpression of Hsp110 is sufficient to prevent endogenous α-synuclein templating and spread following injection of aggregated α-synuclein seeds into brain, supporting a role for Hsp110 in the prevention and/or disaggregation of α-synuclein pathology.

Structural and mechanistic insights into Hsp104 function revealed by synchrotron X-ray footprinting.

Hsp104 is a hexameric AAA+ ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5'-O-(thiotriphosphate)). Comparing side-chain solvent accessibilities between these three states illuminated aspects of Hsp104 structure and guided design of Hsp104 variants to probe the disaggregase mechanism in vitro and in vivo We established that Hsp104 hexamers switch from a more-solvated state in ADP to a less-solvated state in ATPγS, consistent with switching from an open spiral to a closed ring visualized by cryo-EM. We pinpointed critical N-terminal domain (NTD), NTD-nucleotide-binding domain 1 (NBD1) linker, NBD1, and middle domain (MD) residues that enable intrinsic disaggregase activity and Hsp70 collaboration. We uncovered NTD residues in the loop between helices A1 and A2 that can be substituted to enhance disaggregase activity. We elucidated a novel potentiated Hsp104 MD variant, Hsp104-RYD, which suppresses α-synuclein, fused in sarcoma (FUS), and TDP-43 toxicity. We disambiguated a secondary pore-loop in NBD1, which collaborates with the NTD and NBD1 tyrosine-bearing pore-loop to drive protein disaggregation. Finally, we defined Leu-601 in NBD2 as crucial for Hsp104 hexamerization. Collectively, our findings unveil new facets of Hsp104 structure and mechanism. They also connect regions undergoing large changes in solvation to functionality, which could have profound implications for protein engineering.

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding.

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp-substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp-substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.

ATP hydrolysis-coupled peptide translocation mechanism of Mycobacterium tuberculosis ClpB.

The protein disaggregase ClpB hexamer is conserved across evolution and has two AAA+-type nucleotide-binding domains, NBD1 and NBD2, in each protomer. In M. tuberculosis (Mtb), ClpB facilitates asymmetric distribution of protein aggregates during cell division to help the pathogen survive and persist within the host, but a mechanistic understanding has been lacking. Here we report cryo-EM structures at 3.8- to 3.9-Šresolution of Mtb ClpB bound to a model substrate, casein, in the presence of the weakly hydrolyzable ATP mimic adenosine 5'-[γ-thio]triphosphate. Mtb ClpB existed in solution in two closed-ring conformations, conformers 1 and 2. In both conformers, the 12 pore-loops on the 12 NTDs of the six protomers (P1-P6) were arranged similarly to a staircase around the bound peptide. Conformer 1 is a low-affinity state in which three of the 12 pore-loops (the protomer P1 NBD1 and NBD2 loops and the protomer P2 NBD1 loop) are not engaged with peptide. Conformer 2 is a high-affinity state because only one pore-loop (the protomer P2 NBD1 loop) is not engaged with the peptide. The resolution of the two conformations, along with their bound substrate peptides and nucleotides, enabled us to propose a nucleotide-driven peptide translocation mechanism of a bacterial ClpB that is largely consistent with several recent unfoldase structures, in particular with the eukaryotic Hsp104. However, whereas Hsp104's two NBDs move in opposing directions during one step of peptide translocation, in Mtb ClpB the two NBDs move only in the direction of translocation.

Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase.

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.

The molecular language of membraneless organelles.

Eukaryotic cells organize their intracellular components into organelles that can be membrane-bound or membraneless. A large number of membraneless organelles, including nucleoli, Cajal bodies, P-bodies, and stress granules, exist as liquid droplets within the cell and arise from the condensation of cellular material in a process termed liquid-liquid phase separation (LLPS). Beyond a mere organizational tool, concentrating cellular components into membraneless organelles tunes biochemical reactions and improves cellular fitness during stress. In this review, we provide an overview of the molecular underpinnings of the formation and regulation of these membraneless organelles. This molecular understanding explains emergent properties of these membraneless organelles and shines new light on neurodegenerative diseases, which may originate from disturbances in LLPS and membraneless organelles.

Biochemical characterization of ClpB3, a chloroplastic disaggregase from Arabidopsis thaliana.

KEY MESSAGE: The first biochemical characterization of a chloroplastic disaggregase is reported (Arabidopsis thaliana ClpB3). ClpB3 oligomerizes into active hexamers that resolubilize aggregated substrates using ATP and without the aid of partners. Disaggregases from the Hsp100/Clp family are a type of molecular chaperones involved in disassembling protein aggregates. Plant cells are uniquely endowed with ClpB proteins in the cytosol, mitochondria and chloroplasts. Chloroplastic ClpB proteins have been implicated in key processes like the unfolded protein response; however, they have not been studied in detail. In this study, we explored the biochemical properties of a chloroplastic ClpB disaggregase, in particular, ClpB3 from A. thaliana. ClpB3 was produced recombinantly in Escherichia coli and affinity-purified to near homogeneity. ClpB3 forms a hexameric complex in the presence of MgATP and displays intrinsic ATPase activity. We demonstrate that ClpB3 has ATPase activity in a wide range of pH and temperature values and is particularly resistant to heat. ClpB3 specifically targets unstructured polypeptides and mediates the reactivation of heat-denatured model substrates without the aid of the Hsp70 system. Overall, this work represents the first in-depth biochemical description of a ClpB protein from plants and strongly supports its role as the putative disaggregase chaperone in chloroplasts.

Loss of mitochondrial ClpP, Lonp1, and Tfam triggers transcriptional induction of Rnf213, a susceptibility factor for moyamoya disease.

Human RNF213, which encodes the protein mysterin, is a known susceptibility gene for moyamoya disease (MMD), a cerebrovascular condition with occlusive lesions and compensatory angiogenesis. Mysterin mutations, together with exposure to environmental trigger factors, lead to an elevated stroke risk since childhood. Mysterin is induced during cell stress, to function as cytosolic AAA+ ATPase and ubiquitylation enzyme. Little knowledge exists, in which context mysterin is needed. Here, we found that genetic ablation of several mitochondrial matrix factors, such as the peptidase ClpP, the transcription factor Tfam, as well as the peptidase and AAA+ ATPase Lonp1, potently induces Rnf213 transcript expression in various organs, in parallel with other components of the innate immune system. Mostly in mouse fibroblasts and human endothelial cells, the Rnf213 levels showed prominent upregulation upon Poly(I:C)-triggered TLR3-mediated responses to dsRNA toxicity, as well as upon interferon gamma treatment. Only partial suppression of Rnf213 induction was achieved by C16 as an antagonist of PKR (dsRNA-dependent protein kinase). Since dysfunctional mitochondria were recently reported to release immune-stimulatory dsRNA into the cytosol, our results suggest that mysterin becomes relevant when mitochondrial dysfunction or infections have triggered RNA-dependent inflammation. Thus, MMD has similarities with vasculopathies that involve altered nucleotide processing, such as Aicardi-Goutières syndrome or systemic lupus erythematosus. Furthermore, in MMD, the low penetrance of RNF213 mutations might be modified by dysfunctions in mitochondria or the TLR3 pathway.

Amyloid assembly and disassembly.

Amyloid fibrils are protein homopolymers that adopt diverse cross-ß conformations. Some amyloid fibrils are associated with the pathogenesis of devastating neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Conversely, functional amyloids play beneficial roles in melanosome biogenesis, long-term memory formation and release of peptide hormones. Here, we showcase advances in our understanding of amyloid assembly and structure, and how distinct amyloid strains formed by the same protein can cause distinct neurodegenerative diseases. We discuss how mutant steric zippers promote deleterious amyloidogenesis and aberrant liquid-to-gel phase transitions. We also highlight effective strategies to combat amyloidogenesis and related toxicity, including: (1) small-molecule drugs (e.g. tafamidis) to inhibit amyloid formation or (2) stimulate amyloid degradation by the proteasome and autophagy, and (3) protein disaggregases that disassemble toxic amyloid and soluble oligomers. We anticipate that these advances will inspire therapeutics for several fatal neurodegenerative diseases.

The Complex Phosphorylation Patterns that Regulate the Activity of Hsp70 and Its Cochaperones.

Proteins must fold into their native structure and maintain it during their lifespan to display the desired activity. To ensure proper folding and stability, and avoid generation of misfolded conformations that can be potentially cytotoxic, cells synthesize a wide variety of molecular chaperones that assist folding of other proteins and avoid their aggregation, which unfortunately is unavoidable under acute stress conditions. A protein machinery in metazoa, composed of representatives of the Hsp70, Hsp40, and Hsp110 chaperone families, can reactivate protein aggregates. We revised herein the phosphorylation sites found so far in members of these chaperone families and the functional consequences associated with some of them. We also discuss how phosphorylation might regulate the chaperone activity and the interaction of human Hsp70 with its accessory and client proteins. Finally, we present the information that would be necessary to decrypt the effect that post-translational modifications, and especially phosphorylation, could have on the biological activity of the Hsp70 system, known as the "chaperone code".