Phase separation of the mammalian prion protein: Physiological and pathological perspectives.

Abnormal phase transitions have been implicated in the occurrence of proteinopathies. Disordered proteins with nucleic acidbinding ability drive the formation of reversible micron-sized condensates capable of controlling nucleic acid processing/transport. This mechanism, achieved via liquid-liquid phase separation (LLPS), underlies the formation of long-studied membraneless organelles (e.g., nucleolus) and various transient condensates formed by driver proteins. The prion protein (PrP) is not a classical nucleic acid-binding protein. However, it binds nucleic acids with high affinity, undergoes nucleocytoplasmic shuttling, contains a long intrinsically disordered region rich in glycines and evenly spaced aromatic residues, among other biochemical/biophysical properties of bona fide drivers of phase transitions. Because of this, our group and others have characterized LLPS of recombinant PrP. In vitro phase separation of PrP is modulated by nucleic acid aptamers, and depending on the aptamer conformation, the liquid droplets evolve to solid-like species. Herein, we discuss recent studies and previous evidence supporting PrP phase transitions. We focus on the central role of LLPS related to PrP physiology and pathology, with a special emphasis on the interaction of PrP with different ligands, such as proteins and nucleic acids, which can play a role in prion disease pathogenesis. Finally, we comment on therapeutic strategies directed at the non-functional phase separation that could potentially tackle prion diseases or other protein misfolding disorders.

In Vitro Characterization of Protein:Nucleic Acid Liquid-Liquid Phase Separation by Microscopy Methods and Nanoparticle Tracking Analysis.

Uncontrolled assembly/disassembly of physiologically formed liquid condensates is linked to irreversible aggregation. Hence, the quest for understanding protein-misfolding disease mechanism might lie in the studies of protein:nucleic acid coacervation. Several proteins with intrinsically disordered regions as well as nucleic acids undergo phase separation in the cellular context, and this process is key to physiological signaling and is related to pathologies. Phase separation is reproducible in vitro by mixing the target recombinant protein with specific nucleic acids at various stoichiometric ratios and then examined by microscopy and nanotracking methods presented herein. We describe protocols to qualitatively assess hallmarks of protein-rich condensates, characterize their structure using intrinsic and extrinsic dyes, quantify them, and analyze their morphology over time. Analysis by nanoparticle tracking provides information on the concentration and diameter of high-order protein oligomers formed in the presence of nucleic acid. Using the model protein (globular domain of recombinant murine PrP) and DNA aptamers (high-affinity oligonucleotides with 25 nucleotides in length), we provide examples of a systematic screening of liquid-liquid phase separation in vitro.

Cerebral dopamine neurotrophic factor reduces α-synuclein aggregation and propagation and alleviates behavioral alterations in vivo.

A molecular hallmark in Parkinson's disease (PD) pathogenesis are α-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of α-synuclein. Here, we investigated the effects of CDNF on α-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with α-synuclein with a KD = 23 ± 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of α-synuclein fibrils and induces the formation of insoluble phosphorylated α-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with α-synuclein fibrils, though it did not reduce the number of phosphorylated α-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with α-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.

The 1H, 15N, and 13C resonance assignments of the N-terminal domain of the nucleocapsid protein from the Middle East respiratory syndrome coronavirus.

During the past 17 years, the coronaviruses have become a global public emergency, with the first appearance in 2012 in Saudi Arabia of the Middle East respiratory syndrome. Among the structural proteins encoded in the viral genome, the nucleocapsid protein is the most abundant in infected cells. It is a multifunctional phosphoprotein involved in the capsid formation, in the modulation and regulation of the viral life cycle. The N-terminal domain of N protein specifically interacts with transcriptional regulatory sequence (TRS) and is involved in the discontinuous transcription through the melting activity of double-stranded TRS (dsTRS).

Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2.

The hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2 (EcA2), performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. The L-Asp present in Aginasa formulation was found to provide to EcA2 a resistance to in vitro proteolysis. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. Ion-mobility spectrometry-mass spectrometry reveals two conformers for the monomeric EcA2, and that monomeric species has sufficient capacity for selective binding to L-Asp and L-Glu. The N-terminal loop of the EcA2 present in Leuginase, which is part of the active site is disordered, but it gets ordered in the presence of L-Asp, while L-Glu only does so to a limited extent. These data provide new insights on the mechanistic of ligand recognition by EcA2, and the impact of formulation in its conformational diversity landscape.

Phase Separation and Disorder-to-Order Transition of Human Brain Expressed X-Linked 3 (hBEX3) in the Presence of Small Fragments of tRNA.

Brain Expressed X-linked (BEX) protein family consists of five members in humans and is highly expressed during neuronal development. They are known to participate in cell cycle and in signaling pathways involved in neurodegeneration and cancer. BEX3 possess a conserved leucine-rich nuclear export signal and experimental data confirmed BEX3 nucleocytoplasmic shuttling. Previous data revealed that mouse BEX3 auto-associates in an oligomer rich in intrinsic disorder. In this work, we show that human BEX3 (hBEX3) has well-defined three-dimensional structure in the presence of small fragments of tRNA (tRFs). Conversely, the nucleic acids-free purified hBEX3 presented disordered structure. Small-angle X-ray scattering data revealed that in the presence of tRFs, hBEX3 adopts compact globular fold, which is very distinct from the elongated high-order oligomer formed by the pure protein. Furthermore, microscopy showed that hBEX3 undergoes condensation in micron-sized protein-rich droplets in vitro. In the presence of tRFs, biomolecular condensates were smaller and in higher number, showing acridine orange green fluorescence emission, which corroborated with the presence of base-paired nucleic acids. Additionally, we found that over time hBEX3 transits from liquid condensates to aggregates that are reversible upon temperature increment and dissolved by 1,6-hexanediol. hBEX3 assemblies display different morphology in the presence of the tRFs that seems to protect from amyloid formation. Collectively, our findings support a role for tRFs in hBEX3 disorder-to-order transition and modulation of phase transitions. Moreover, hBEX3 aggregation-prone features and the specificity in interaction with tRNA fragments advocate paramount importance toward understanding BEX family involvement in neurodevelopment and cell death.

Liquid-liquid phase separation and fibrillation of the prion protein modulated by a high-affinity DNA aptamer.

Structural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies (TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid-liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25-mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP90-231) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP90-231 and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP90-231 phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration-dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs.

1H, 13C and 15N chemical shift assignment of lissencephaly-1 homology (LisH) domain homodimer of human two-hybrid-associated protein 1 with RanBPM (Twa1).

The CTLH complex is a large, highly conserved eukaryotic complex composed of eight proteins that has been associated to several cellular functions, more often described as an E3 ubiquitin ligase complex involved in protein degradation through ubiquitination but also via vacuole-dependent degradation. A common feature observed in several components of this complex is the presence of the domains lissencephaly-1 homology (LisH) and C-terminal to LisH (CTLH). The LisH domain is found in several proteins involved in chromosome segregation, microtubule dynamics, and cell migration. Also, this domain participates in protein dimerization, besides affecting protein half-life, and influencing in specific cellular localization. Among the proteins found in the CTLH complex, Twa1 (Two-hybrid-associated protein 1 with RanBPM), also known as Gid8 (glucose-induced degradation protein 8 homolog) is the smallest, being a good model for structural studies by NMR. In this work we report the chemical shift assignments of the homodimeric LisH domain of Twa1, as a first step to determine its solution structure.

Biophysical Studies on BEX3, the p75NTR-Associated Cell Death Executor, Reveal a High-Order Oligomer with Partially Folded Regions.

BEX3 (Brain Expressed X-linked protein 3) is a member of a mammal-specific placental protein family. Several studies have found the BEX proteins to be associated with neurodegeneration, the cell cycle and cancer. BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. The pro-apoptotic activity of BEX3 in association with a number of additional proteins has been widely supported; however, to the best of our knowledge, very limited data are available on the conformation of any of the members of the BEX family. In this study, we structurally characterized BEX3 using biophysical experimental data. Small angle X-ray scattering and atomic force microscopy revealed that BEX3 forms a specific higher-order oligomer that is consistent with a globular molecule. Solution nuclear magnetic resonance, partial proteinase K digestion, circular dichroism spectroscopy, and fluorescence techniques that were performed on the recombinant protein indicated that the structure of BEX3 is composed of approximately 31% α-helix and 20% ß-strand, contains partially folded regions near the N- and C-termini, and a core which is proteolysis-resistant around residues 55-120. The self-oligomerization of BEX3 has been previously reported in cell culture and is consistent with our in vitro data.

The Solution Structure and Dynamics of Full-length Human Cerebral Dopamine Neurotrophic Factor and Its Neuroprotective Role against α-Synuclein Oligomers.

Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.

(1)H-, (13)C- and (15)N-NMR assignment of the N-terminal domain of human cerebral dopamine neurotrophic factor (CDNF).

Parkinson's disease (PD) is a neurodegenerative disorder that is caused by the death of midbrain dopaminergic neurons. Current therapies for PD do not halt the neurodegeneration nor repair the affected neurons. Therefore, search for novel neurotrophic factors (NTF) for midbrain dopaminergic neurons, which could be used in novel therapeutic approaches, is highly wanted. In 2007, a potent NTF for dopaminergic neurons was described as the conserved dopamine neurotrophic factor (CDNF). Single doses of this protein protect and restore dopaminergic neurons in experimental models of PD. CDNF has two domains; an N-terminal saposin-like domain, which may bind to membranes; and a presumably intrinsically unstructured C-terminal which contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus reduce the endoplasmic reticulum stress caused by incorrectly folded proteins. We show for the first time the nuclear magnetic resonance assignment of N-terminal domain of recombinant CDNF (residues 1-105) by solution 2D and 3D NMR spectroscopy. We were able to obtain a nearly complete resonance assignment, which is the first step toward the solution structure determination of this neurotrophic factor.

Heparin binding by murine recombinant prion protein leads to transient aggregation and formation of RNA-resistant species.

The conversion of cellular prion protein (PrP(C)) into the pathological conformer PrP(Sc) requires contact between both isoforms and probably also requires a cellular factor, such as a nucleic acid or a glycosaminoglycan (GAG). Little is known about the structural features implicit in the GAG-PrP interaction. In the present work, light scattering, fluorescence, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy were used to describe the chemical and physical properties of the murine recombinant PrP 23-231 interaction with low molecular weight heparin (LMWHep) at pH 7.4 and 5.5. LMWHep interacts with rPrP 23-231, thereby inducing transient aggregation. The interaction between murine rPrP and heparin at pH 5.5 had a stoichiometry of 2:1 (LMWHep:rPrP 23-231), in contrast to a 1:1 binding ratio at pH 7.4. At binding equilibrium, NMR spectra showed that rPrP complexed with LMWHep had the same general fold as that of the free protein, even though the binding can be indicated by significant changes in few residues of the C-terminal domain, especially at pH 5.5. Notably, the soluble LMWHep:rPrP complex prevented RNA-induced aggregation. We also investigated the interaction between LMWHep and the deletion mutants rPrP Δ51-90 and Δ32-121. Heparin did not bind these constructs at pH 7.4 but was able to interact at pH 5.5, indicating that this glycosaminoglycan binds the octapeptide repeat region at pH 7.4 but can also bind other regions of the protein at pH 5.5. The interaction at pH 5.5 was dependent on histidine residues of the murine rPrP 23-231. Depending on the cellular milieu, the PrP may expose different regions that can bind GAG. These results shed light on the role of GAGs in PrP conversion. The transient aggregation of PrP may explain why some GAGs have been reported to induce the conversion into the misfolded, scrapie conformation, whereas others are thought to protect against conversion. The acquired resistance of the complex against RNA-induced aggregation explains some of the unique properties of the PrP interaction with GAGs.

Mapping the interactions between a major pollen allergen and human IgE antibodies.

The interaction of specific IgE antibodies with allergens is a key event in the induction of allergic symptoms, thus representing an important target for therapeutic interventions in Type I allergies. We report here the solution NMR structure of Art v 1, the major mugwort pollen allergen. Art v 1 is the first protein structure with an allergenic defensin fold linked to a polyproline domain, which has not been identified in any reported allergen structure in the PDB. Moreover, the direct interaction of polyclonal IgE antibodies from an allergic patient has been mapped on the surface of an allergen for the first time. The data presented herein provide the basis for the design of tools for safe and effective vaccination against mugwort pollen allergy.

Sequence-specific 1H, 15N and 13C resonance assignments of Art v 1: a proline-rich allergen of Artemisia vulgaris pollen.

Art v 1 is the major allergen of Artemisia vulgaris. The IgE raised against Art v 1 not only can cross-react with other proteins from the Asteraceae family members but also with components of various forms of food. Art v 1 is an important target for immunotherapy strategies, including vaccination with hypoallergenic derivatives or chimeras. We report the (1)H, (13)C, and (15)N resonance assignments of the recombinant Art v 1 and identification of secondary structures based on (13)C chemical shifts.

Prion protein complexed to N2a cellular RNAs through its N-terminal domain forms aggregates and is toxic to murine neuroblastoma cells.

Conversion of the cellular prion protein (PrP(C)) into its altered conformation, PrP(Sc), is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher beta-sheet content and characteristics similar to those of PrP(Sc). RNA molecules can also interact with PrP and potentially modulate PrP(C) to PrP(Sc) conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP(C)-->PrP(Sc) conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.

Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus.

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.

Structure of the Ebola fusion peptide in a membrane-mimetic environment and the interaction with lipid rafts.

The fusion peptide EBO(16) (GAAIGLAWIPYFGPAA) comprises the fusion domain of an internal sequence located in the envelope fusion glycoprotein (GP2) of the Ebola virus. This region interacts with the cellular membrane of the host and leads to membrane fusion. To gain insight into the mechanism of the peptide-membrane interaction and fusion, insertion of the peptide was modeled by experiments in which the tryptophan fluorescence and (1)H NMR were monitored in the presence of sodium dodecyl sulfate micelles or in the presence of detergent-resistant membrane fractions. In the presence of SDS micelles, EBO(16) undergoes a random coil-helix transition, showing a tendency to self-associate. The three-dimensional structure displays a 3(10)-helix in the central part of molecule, similar to the fusion peptides of many known membrane fusion proteins. Our results also reveal that EBO(16) can interact with detergent-resistant membrane fractions and strongly suggest that Trp-8 and Phe-12 are important for structure maintenance within the membrane bilayer. Replacement of tryptophan 8 with alanine (W8A) resulted in dramatic loss of helical structure, proving the importance of the aromatic ring in stabilizing the helix. Molecular dynamics studies of the interaction between the peptide and the target membrane also corroborated the crucial participation of these aromatic residues. The aromatic-aromatic interaction may provide a mechanism for the free energy coupling between random coil-helical transition and membrane anchoring. Our data shed light on the structural "domains" of fusion peptides and provide a clue for the development of a drug that might block the early steps of viral infection.

NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase.

A screen of the human cancer genome anatomy project (CGAP) database was performed to search for new proteins involved in tumorigenesis. The resulting hits were further screened for recombinant expression, solubility and protein aggregation, which led to the identification of the previously unknown human cancer-related (HCR) protein encoded by the mRNA NM_032324 as a target for structure determination by NMR. The three-dimensional structure of the protein in its complex with ATPgammaS forms a three-layered alpha/beta sandwich, with a central nine-stranded beta-sheet surrounded by five alpha-helices. Sequence and three-dimensional structure comparisons with AAA+ ATPases revealed the presence of Walker A (GPPGVGKT) and Walker B (VCVIDEIG) motifs. Using 1D (31)P-NMR spectroscopy and a coupled enzymatic assay for the determination of inorganic phosphate, we showed that the purified recombinant protein is active as a non-specific nucleoside triphosphatase, with k(cat)=7.6x10(-3) s(-1). The structural basis for the enzymatic activity of HCR-NTPase was further characterized by site-directed mutagenesis of the Walker B motif, which further contributes to making the HCR-NTPase an attractive new target for further biochemical characterization in the context of its presumed role in human tumorigenesis.

Novel beta-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus.

The nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus has 179 residues and is the N-terminal cleavage product of the viral replicase polyprotein that mediates RNA replication and processing. The specific function of nsp1 is not known. Here we report the nuclear magnetic resonance structure of the nsp1 segment from residue 13 to 128, which represents a novel alpha/beta-fold formed by a mixed parallel/antiparallel six-stranded beta-barrel, an alpha-helix covering one opening of the barrel, and a 3(10)-helix alongside the barrel. We further characterized the full-length 179-residue protein and show that the polypeptide segments of residues 1 to 12 and 129 to 179 are flexibly disordered. The structure is analyzed in a search for possible correlations with the recently reported activity of nsp1 in the degradation of mRNA.