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Prion diseases are characterized by prion protein (PrP) transmissible aggregation and toxicity in the brain. The physiological function of PrP seems related to sequestering and internalization of redox-active Cu2+. It is unclear whether Cu2+contributes to PrP aggregation, recently shown to be mediated by PrP condensation. We investigated the role of Cu2+and oxidation in PrP condensation and aggregation using multiple biophysical and biochemical methods. We find that Cu2+promotes PrP condensation at the cell surface andin vitrothrough co-partitioning. Molecularly, Cu2+inhibited PrP β-structure and hydrophobic residues exposure. Oxidation, induced by H2O2, triggered liquid-to-solid transition of PrP:Cu2+condensates and promoted amyloid-like PrP aggregation. In cells, overexpression of PrPCinitially protected against Cu2+cytotoxicity but led to PrPCaggregation upon extended copper exposure. Our data suggest that PrP condensates function as a buffer for copper that prevent copper toxicity but can transition into PrP aggregation at prolonged oxidative stress.

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.

Alzheimer's disease (AD) is related to the presence of extracellular aggregated amyloid-β peptide (Aβ), which binds copper(II) with high affinity in its N-terminal region. In this sense, two new 1-methylimidazole-containing N-acylhydrazonic metallophores, namely, X1TMP and X1Benz, were synthesized as hydrochlorides and characterized. The compound X1TMP contains the 3,4,5-trimethoxybenzoyl moiety present in the structure of mescaline, a natural hallucinogenic protoalkaloid that occurs in some species of cacti. Single crystals of X1Benz, the unsubstituted derivative of X1TMP, were obtained. The experimental partition coefficients of both compounds were determined, as well as their apparent affinity for Cu

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.

After four decades of prion protein research, the pressing questions in the literature remain similar to the common existential dilemmas. Who am I? Some structural characteristics of the cellular prion protein (PrP

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.

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are neurodegenerative disorders which affect mammals, including the human species, and arise after the conversion of the monomeric cellular prion protein (PrPC) into the aggregated scrapie form (PrPSc). There is no therapy to treat TSEs and the identification of compounds that bind PrPC, preventing its conversion into PrPSc, is a viable therapeutic strategy. We designed and synthesized six novel trimethoxy-benzamide compounds as anti-prion drug candidates. Molecular docking analyses predicted that all the derivatives bind to a hotspot region located in the PrP globular domain with very similar spatial orientation and interaction mode. Although none of the analogs inhibited in vitro-aggregation of recombinant PrP (rPrP) in a cell-free conversion assay, the RT-QuIC, compound 8a accelerated rPrP conversion into PrPSc-like species. STD-NMR and ITC analyses indicated that both 8a and 8b bind to rPrP90–231. These analogs were toxic to PrPSc-infected cell lines, hence we could not assess their anti-prion activity by using this cellular approach, although this toxicity was cell line-dependent. These results point out that the 4-amino-quinoline trimethoxy-benzamide scaffold described herein represents a novel chemical pattern useful as a starting point for future structural optimization in the design of PrP ligands with improved affinity and safety profiles.

Recent studies have proposed that nucleic acids act as potential cofactors for protein aggregation and prionogenesis. By means of sedimentation, transmission electron microscopy, circular dichroism, static and dynamic light scattering, we have studied how RNA can influence the aggregation of the murine recombinant prion protein (rPrP). We find that RNA, independent of its sequence, source and size, modulates rPrP aggregation in a bimodal fashion, affecting both the extent and the rate of rPrP aggregation in a concentration dependent manner. Analogous to RNA-induced liquid-liquid phase transitions observed for other proteins implicated in neurodegenerative diseases, high protein to RNA ratios stimulate rPrP aggregation, while low ratios suppress it. However, the latter scenario also promotes formation of soluble oligomeric aggregates capable of seeding de novo rPrP aggregation. Furthermore, RNA co-aggregates with rPrP and thereby gains partial protection from RNase digestion. Our results also indicate that rPrP interacts with the RNAs with its N-terminus. In summary, this study elucidates the proposed adjuvant role of RNA in prion protein aggregation and propagation, and thus advocates an auxiliary role of the nucleic acids in protein aggregation in general. Manuscript title: Direct involvement of RNA in mammalian prion protein aggregation: Involvement of RNA in rPrP aggregation

A new series of 1,4-disubstituted-1,2,3-triazole derivatives were synthesized through the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (Click chemistry) and their inhibitory activities were evaluated against different human glioblastoma (GBM) cell lines, including highly drug-resistant human cell lines GBM02, GBM95. The most effective compounds were 9d, containing the methylenoxy moiety linked to triazole and the tosyl-hydrazone group, and the symmetrical bis-triazole 10a, also containing methylenoxy moiety linked to triazole. Single crystal X-ray diffraction analysis was employed for structural elucidation of compound 9d. In silico analyses of physicochemical, pharmacokinetic, and toxicological properties suggest that compounds 8a, 8b, 8c, 9d, and 10a are potential candidates for central nervous system-acting drugs.

Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF). The mechanism of the formation of the aggregates in the nucleus remains uncertain. The present study demonstrated that the DNA-binding domain of p53 (p53C) underwent phase separation (PS) on the pathway to aggregation under various conditions. p53C phase separated in the presence of the crowding agent polyethylene glycol (PEG). Similarly, mutant p53C (M237I and R249S) underwent PS; however, the process evolved to a solid-like phase transition faster than that in the case of wild-type p53C. The data obtained by microscopy of live cells indicated that transfection of mutant full-length p53 into the cells tended to result in PS and phase transition (PT) in the nuclear compartments, which are likely the cause of the GoF effects. Fluorescence recovery after photobleaching (FRAP) experiments revealed liquid characteristics of the condensates in the nucleus. Mutant p53 tended to undergo gel- and solid-like phase transitions in the nucleus and in nuclear bodies demonstrated by slow and incomplete recovery of fluorescence after photobleaching. Polyanions, such as heparin and RNA, were able to modulate PS and PT in vitro. Heparin apparently stabilized the condensates in a gel-like state, and RNA apparently induced a solid-like state of the protein even in the absence of PEG. Conditions that destabilize p53C into a molten globule conformation also produced liquid droplets in the absence of crowding. The disordered transactivation domain (TAD) modulated both phase separation and amyloid aggregation. In summary, our data provide mechanistic insight into the formation of p53 condensates and conditions that may result in the formation of aggregated structures, such as mutant amyloid oligomers, in cancer. The pathway of mutant p53 from liquid droplets to gel-like and solid-like (amyloid) species may be a suitable target for anticancer therapy., Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF).

After four decades of prion protein research, the pressing questions in the literature remain similar to the common existential dilemmas. Who am I? Some structural characteristics of the cellular prion protein (PrPC) and scrapie PrP (PrPSc) remain unknown: there are no high-resolution atomic structures for either full-length endogenous human PrPC or isolated infectious PrPSc particles. Why am I here? It is not known why PrPC and PrPSc are found in specific cellular compartments such as the nucleus; while the physiological functions of PrPC are still being uncovered, the misfolding site remains obscure. Where am I going? The subcellular distribution of PrPC and PrPSc is wide (reported in 10 different locations in the cell). This complexity is further exacerbated by the eight different PrP fragments yielded from conserved proteolytic cleavages and by reversible post-translational modifications, such as glycosylation, phosphorylation, and ubiquitination. Moreover, about 55 pathological mutations and 16 polymorphisms on the PrP gene (PRNP) have been described. Prion diseases also share unique, challenging features: strain phenomenon (associated with the heterogeneity of PrPSc conformations) and the possible transmissibility between species, factors which contribute to PrP undruggability. However, two recent concepts in biochemistry—intrinsically disordered proteins and phase transitions—may shed light on the molecular basis of PrP's role in physiology and disease.

Although prion protein fibrils can have either parallel-in-register intermolecular β-sheet (PIRIBS) or, probably, β-solenoid architectures, the plausibility of PIRIBS architectures for the usually glycosylated natural prion strains has been questioned based the expectation that such glycans would not fit if stacked in-register on each monomer within a fibril. To directly assess this issue, we have added N-linked glycans to a recently reported cryo-electron microscopy-based human prion protein amyloid model with a PIRIBS architecture and performed in silico molecular dynamics studies to determine if the glycans can fit. Our results show that triantennary glycans can be sterically accommodated in-register on both N-linked glycosylation sites of each monomer. Additional simulations with an artificially mutated β-solenoid model confirmed that glycans can be accommodated when aligned with ∼4.8 A spacing on every rung of a fibril. Altogether, we conclude that steric intermolecular clashes between glycans do not, in themselves, preclude PIRIBS architectures for prions.

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, arise from the structural conversion of the monomeric, cellular prion protein (PrPC) into its multimeric scrapie form (PrPSc). These pathologies comprise a group of intractable, rapidly evolving neurodegenerative diseases. Currently, a definitive diagnosis of TSE relies on the detection of PrPSc and/or the identification of pathognomonic histological features in brain tissue samples, which are usually obtained postmortem or, in rare cases, by brain biopsy (antemortem). Over the past two decades, several paraclinical tests for antemortem diagnosis have been developed to preclude the need for brain samples. Some of these alternative methods have been validated and can provide a probable diagnosis when combined with clinical evaluation. Paraclinical tests include in vitro cell-free conversion techniques, such as the real-time quaking-induced conversion (RT-QuIC), as well as immunoassays, electroencephalography (EEG), and brain bioimaging methods, such as magnetic resonance imaging (MRI), whose importance has increased over the years. PrPSc is the main biomarker in TSEs, and the RT-QuIC assay stands out for its ability to detect PrPSc in cerebrospinal fluid (CSF), olfactory mucosa, and dermatome skin samples with high sensitivity and specificity. Other biochemical biomarkers are the proteins 14-3-3, tau, neuron-specific enolase (NSE), astroglial protein S100B, α-synuclein, and neurofilament light chain protein (NFL), but they are not specific for TSEs. This paper reviews the techniques employed for definite diagnosis, as well as the clinical and paraclinical methods for possible and probable diagnosis, both those in use currently and those no longer employed. We also discuss current criteria, challenges, and perspectives for TSE diagnosis. An early and accurate diagnosis may allow earlier implementation of strategies to delay or stop disease progression.

Liquid-liquid phase separation (LLPS) and phase transitions (PT) of proteins, which include the formation of gel- and solid-like species, have been characterized as physical processes related to the pathology of conformational diseases. Nucleic acid (NA)-binding proteins related to neurodegenerative disorders and cancer were shown by us and others to experience PT modulated by different NAs. Herein, we discuss recent work on phase separation and phase transitions of two amyloidogenic proteins, i.e. the prion protein (PrP) and p53, which undergo conformational changes and aggregate upon NA interaction. The role of different NAs in these processes is discussed to shed light on the relevance of PSs and PTs for both the functional and pathological roles of these mammalian proteins.

Conversion of the cellular prion protein (PrPC) into the scrapie form (PrPSc) is the leading step to the development of transmissible spongiform encephalopathies (TSEs), still incurable neurodegenerative disorders. Interaction of PrPC with cellular and synthetic ligands that induce formation of scrapie-like conformations has been deeply investigated in vitro. Different nucleic acid (NA) sequences bind PrP and convert it to β-sheet-rich or unfolded species; among such NAs, a 21-mer double-stranded DNA, D67, was shown to induce formation of PrP aggregates that were cytotoxic. However, in vivo effects of these PrP-DNA complexes were not explored. Herein, aggregates of recombinant full-length PrP (rPrP23-231) induced by interaction with the D67 aptamer were inoculated into the lateral ventricle of Swiss mice and acute effects were investigated. The aggregates had no influence on emotional, locomotor and motor behavior of mice. In contrast, mice developed cognitive impairment and hippocampal synapse loss, which was accompanied by intense activation of glial cells in this brain region. Our results suggest that the i.c.v. injection of rPrP:D67 aggregates is an interesting model to study the neurotoxicity of aggregated PrP in vivo, and that glial cell activation may be an important step for behavioral and cognitive dysfunction in prion diseases.

Liquid-liquid phase separation (LLPS) and phase transition (LLPT) of proteins and nucleic acids have emerged as a new paradigm in cell biology. Here we will describe the recent findings about LLPS and LLPT, including the molecular and physical determinants leading to their formation, the resulting functions and their implications in cell physiology and disease. Amyloid aggregation is implicated in many neurodegenerative diseases and cancer, and LLPS of proteins involved in these diseases appear to be related to their function in different cell contexts. Amyloid formation would correspond to an irreversible liquid-to-solid transition, as clearly observed in the case of PrP, TDP43, FUS/TLS and tau protein in neurodegenerative pathologies as well as with the mutant tumor suppressor p53 in cancer. Nucleic acids play a modulatory effect on both LLPS and amyloid aggregation. Understanding the molecular events regulating how the demixing process advances to solid-like fibril materials is crucial for the development of novel therapeutic strategies against cancer and neurodegenerative maladies.

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.

Structural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events for the onset of Transmissible Spongiform Encephalopathies (TSEs). Experimental evidences support the role of nucleic acids (NAs) in assisting the protein conversion process. Here, we used the SELEX methodology to identify two 25-mer DNA aptamers against the globular domain of recombinant murine PrP (rPrP90-231), namely A1 and A2. High-affinity binding of A1 and A2 to rPrP was verified by ITC. Aptamers structure was characterized by theoretical predictions, CD, NMR and SAXS, revealing that A1 adopts a hairpin conformation. Aptamer binding caused dynamic aggregation of rPrP90-231, resulting from the ability of rPrP90-231to undergo liquid-liquid phase separation (LLPS). While free rPrP90-231phase separated into large droplets, aptamer binding increased the amount but reduced the size of the condensates. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced transition to an ordered state, suggestive of amyloid formation on the surface of the droplets. Our results describe for the first time PrP:NA interaction leading to LLPS and modulation of this effect depending on NA structure and binding stoichiometry, shedding light on the role of NAs in PrP misfolding and TSEs.

Two commercially available and widely used enzymes, the parent Thermomyces lanuginosus lipase (TLL) and the shuffled phospholipase A1 Lecitase (Lecitase Ultra), were encapsulated in AOT/isooctane reverse micelles and evaluated regarding their structure and activity. Preparations were also tested as effective biocatalysts. Small-angle X-ray scattering (SAXS), electronic paramagnetic resonance (EPR), and fluorescence spectroscopy were the techniques applied to assess the effects of enzyme incorporation to a reverse micellar nanostructure. SAXS analysis showed that the radius of gyration (Rg) changed from 16 to 38 Å, as the water content (w0) increased. Elongated shapes were more commonly observed than spherical shapes after enzyme encapsulation. EPR studies indicated that enzymes do not participate in the interface, being located in the aqueous center. Fluorescence energy transfer showed that TLL is located in the water core, whereas Lecitase Ultra is closer to the interface. Enzymatic activity toward a standard esterification reaction endured after the enzyme was incorporated into the micelles. The activity of TLL for systems with w0 15 showed the highest conversion yield, 38% in 2 h, while the system with w0 10 showed the highest initial velocity, 0.43 μM/min. This last system had a Rg of 19.3 Å, similar to that of the TLL monomer. Lecitase Ultra showed the highest conversion yields in systems with w0 10, 55% in 2 h. However, the initial rate was much lower than that of TLL, suggesting less affinity for the substrates, which is expected since Lecitase Ultra is a phospholipase. In summary, we here used several spectroscopic and scattering techniques to reveal the shape and stability of TTL and Lecitase Ultra encapsulated systems, which allowed the selection of w0 values to provide optimized enzymatic activity.

A series of 3-substituted-7-aminoalcoxy-coumarin was designed and evaluated as cholinesterase inhibitors and antioxidants. All compounds were effective in inhibiting AChE with potencies in the nanomolar range. The 3-(4-(dimethylamino)phenyl)-7-aminoethoxy-coumarin (6a) was considered a hit, showing good AChE inhibition potency (IC50 = 20 nM) and selectivity (IC50 BuChE/AChE = 354), quite similar to the reference drug donepezil (IC50 = 6 nM; IC50 BuChE/AChE = 365), also presenting antioxidant properties, low citotoxicity and good-predicted ADMET properties. The mode of action (mixed-type) and SAR analysis for this series of compounds were described by means of kinetic and molecular modeling evaluations., Graphical Abstract

Liquid-liquid phase separation (LLPS) and phase transition (LLPT) of proteins and nucleic acids have emerged as a new paradigm in cell biology. Here we will describe the recent findings about LLPS and LLPT, including the molecular and physical determinants leading to their formation, the resulting functions and their implications in cell physiology and disease. Amyloid aggregation is implicated in many neurodegenerative diseases and cancer, and LLPS of proteins involved in these diseases appear to be related to their function in different cell contexts. Amyloid formation would correspond to an irreversible liquid-to-solid transition, as clearly observed in the case of PrP, TDP43, FUS/TLS and tau protein in neurodegenerative pathologies as well as with the mutant tumor suppressor p53 in cancer. Nucleic acids play a modulatory effect on both LLPS and amyloid aggregation. Understanding the molecular events regulating how the demixing process advances to solid-like fibril materials is crucial for the development of novel therapeutic strategies against cancer and neurodegenerative maladies.

Background Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein. Results Pressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods. Conclusions The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein. Electronic supplementary material The online version of this article (10.1186/s12896-018-0486-2) contains supplementary material, which is available to authorized users.

The search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrP C ) conversion into protease-resistant forms (protease-resistant PrP [PrP Res ] or the scrapie form of PrP [PrP Sc ]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identified in vitro to be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrP C mRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP 23–231 ) as the substrate and PrP Sc seeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP 90–231 ), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrP C globular domain. J8 also reduced the fibrillation of mouse rPrP 23–231 seeded with in vitro -produced fibrils. Furthermore, most of the compounds decreased the amount of PrP C on the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrP C content on the cell surface.

Raw data of Figs. 1,2, 4 and 7. Data used for preparation of the Figs. 1, 2, 4 and 7. (DOCX 17 kb)

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative disorders that affect humans and other mammals. The etiologic agents common to these diseases are misfolded conformations of the prion protein (PrP). The molecular mechanisms that trigger the structural conversion of the normal cellular PrP (PrPC) into the pathogenic conformer (PrPSc) are still poorly understood. It is proposed that a molecular cofactor would act as a catalyst, lowering the activation energy of the conversion process, therefore favoring the transition of PrPC to PrPSc. Several in vitro studies have described physical interactions between PrP and different classes of molecules, which might play a role in either PrP physiology or pathology. Among these molecules, nucleic acids (NAs) are highlighted as potential PrP molecular partners. In this context, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology has proven extremely valuable to investigate PrP–NA interactions, due to its ability to select small nucleic acids, also termed aptamers, that bind PrP with high affinity and specificity. Aptamers are single-stranded DNA or RNA oligonucleotides that can be folded into a wide range of structures (from harpins to G-quadruplexes). They are selected from a nucleic acid pool containing a large number (1014–1016) of random sequences of the same size (~20–100 bases). Aptamers stand out because of their potential ability to bind with different affinities to distinct conformations of the same protein target. Therefore, the identification of high-affinity and selective PrP ligands may aid the development of new therapies and diagnostic tools for TSEs. This review will focus on the selection of aptamers targeted against either full-length or truncated forms of PrP, discussing the implications that result from interactions of PrP with NAs, and their potential advances in the studies of prions. We will also provide a critical evaluation, assuming the advantages and drawbacks of the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique in the general field of amyloidogenic proteins.

Mammalian prion proteins (PrPs) that cause transmissible spongiform encephalopathies are misfolded conformations of the host cellular PrP. The misfolded form, the scrapie PrP (PrP(Sc)), can aggregate into amyloid fibrils that progressively accumulate in the brain, evolving to a pathological phenotype. A particular characteristic of PrP(Sc) is to be found as different strains, related to the diversity of conformational states it can adopt. Prion strains are responsible for the multiple phenotypes observed in prion diseases, presenting different incubation times and diverse deposition profiles in the brain. PrP biochemical properties are also strain-dependent, such as different digestion pattern after proteolysis and different stability. Although they have long been studied, strain formation is still a major unsolved issue in prion biology. The recreation of strain-specific conformational features is of fundamental importance to study this unique pathogenic phenomenon. In our recent paper, we described that murine PrP, when expressed in bacteria, forms amyloid inclusion bodies that possess different strain-like characteristics, depending on the PrP construct. Here, we present an extra-view of these data and propose that bacteria might become a successful model to generate preparative amounts of prion strain-specific assemblies for high-resolution structural analysis as well as for addressing the determinants of infectivity and transmissibility.

Background: Proteins have adopted negative design to diminish aggregation. Results: The replacement of Lys-35 by Leu increases the amyloidogenicity of the 26–57 segment of TTR as well as the entire protein. Conclusion: Lys-35 is as a gatekeeper residue in TTR, and its protective effect is suppressed by heparin. Significance: The elucidation of the principles that govern protein aggregation is helpful for the design of strategies against amyloid diseases., Protein aggregation into β-sheet-enriched amyloid fibrils is associated with an increasing number of human disorders. The adoption of such amyloid conformations seems to constitute a generic property of polypeptide chains. Therefore, during evolution, proteins have adopted negative design strategies to diminish their intrinsic propensity to aggregate, including enrichment of gatekeeper charged residues at the flanks of hydrophobic aggregation-prone segments. Wild type transthyretin (TTR) is responsible for senile systemic amyloidosis, and more than 100 mutations in the TTR gene are involved in familial amyloid polyneuropathy. The TTR 26–57 segment bears many of these aggressive amyloidogenic mutations as well as the binding site for heparin. We demonstrate here that Lys-35 acts as a gatekeeper residue in TTR, strongly decreasing its amyloidogenic potential. This protective effect is sequence-specific because Lys-48 does not affect TTR aggregation. Lys-35 is part of the TTR basic heparin-binding motif. This glycosaminoglycan blocks the protective effect of Lys-35, probably by neutralization of its side chain positive charge. A K35L mutation emulates this effect and results in the rapid self-assembly of the TTR 26–57 region into amyloid fibrils. This mutation does not affect the tetrameric protein stability, but it strongly increases its aggregation propensity. Overall, we illustrate how TTR is yet another amyloidogenic protein exploiting negative design to prevent its massive aggregation, and we show how blockage of conserved protective features by endogenous factors or mutations might result in increased disease susceptibility.

The conversion of the prion protein (PrP) into scrapie PrP (PrPSc) is a central event in prion diseases. Several molecules work as cofactors in the conversion process, including glycosaminoglycans (GAGs). GAGs exhibit a paradoxical effect, as they convert PrP into protease-resistant PrP (PrP-res) but also exert protective activity. We compared the stability and aggregation propensity of PrP and the heparin-PrP complex through the application of different in vitro aggregation approaches, including real-time quaking-induced conversion (RT-QuIC). Transmissible spongiform encephalopathy–associated forms from mouse and hamster brain homogenates were used to seed RT-QuIC-induced fibrillization. In our study, interaction between heparin and cellular PrP (PrPC) increased thermal PrP stability, leading to an 8-fold decrease in temperature-induced aggregation. The interaction of low-molecular-weight heparin (LMWHep) with the PrP N- or C-terminal domain affected not only the extent of PrP fibrillization but also its kinetics, lowering the reaction rate constant from 1.04 to 0.29 s−1 and increasing the lag phase from 12 to 19 h in RT-QuIC experiments. Our findings explain the protective effect of heparin in different models of prion and prion-like neurodegenerative diseases and establish the groundwork for the development of therapeutic strategies based on GAGs.—Vieira, T. C. R. G., Cordeiro, Y., Caughey, B., Silva, J. L. Heparin binding confers prion stability and impairs its aggregation.

Protein misfolding disorders (PMDs) refer to a group of diseases related to the misfolding of particular proteins that aggregate and deposit in the cells and tissues of humans and other mammals. The mechanisms that trigger protein misfolding and aggregation are still not fully understood. Increasing experimental evidence indicates that abnormal interactions between PMD-related proteins and nucleic acids (NAs) can induce conformational changes. Here, we discuss these protein–NA interactions and address the role of deoxyribonucleic (DNA) and ribonucleic (RNA) acid molecules in the conformational conversion of different proteins that aggregate in PMDs, such as Alzheimer’s, Parkinson’s, and prion diseases. Studies on the affinity, stability, and specificity of proteins involved in neurodegenerative diseases and NAs are specifically addressed. A landscape of reciprocal effects resulting from the binding of prion proteins, amyloid-β peptides, tau proteins, huntingtin, and α-synuclein are presented here to clarify the possible role of NAs, not only as encoders of genetic information but also in triggering PMDs.

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 °C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 °C are more efficiently dissociated at 2.4 kbar than those produced at 47 °C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (−9 °C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 °C to 50 °C is proportional to the temperature, and a higher yield was obtained at 20 °C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 °C), of their dissociation at HHP (−9 °C) and of eGFP refolding (20 °C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.

Lipases are triacyl glycerol acyl hydrolases, which catalyze hydrolysis of esters, esterification and transesterification reactions, among others. Some of these enzymes have a large hydrophobic pocket covered by an alpha-helical mobile surface loop (the lid). Protein-protein interactions can occur through adsorption of two open lids of individual lipases. We investigated the conformation and oligomeric state of Thermomyces lanuginosus lipase (TLL) in solution by spectroscopic and mass spectrometry techniques. Information about oligomerization of this important industrial enzyme is only available for TLL crystals; therefore, we have done a throughout investigation of the conformation of this lipase in solution. SDS-PAGE and mass spectrometry analysis of size-exclusion chromatography eluted fractions indicated the presence of both monomeric and dimeric populations of TLL. The stability of the enzyme upon thermal and guanidine hydrochloride treatment was examined by circular dichroism and fluorescence emission spectroscopy. Small angle x-ray scattering and ion mobility mass spectrometry analysis revealed that TLL is found as a mixture of monomers and dimers at the assayed concentrations. Although previous x-ray diffraction data showed TLL as a dimer in the crystal (PDB: 1DT3), to our knowledge our report is the first evidencing that TLL co-exists as stable dimeric and monomeric forms in solution.

High hydrostatic pressure (HHP) is a valuable tool to study processes such as protein folding, protein hydration and protein–protein interactions. HHP is a nondestructive technique because it reversibly affects internal cavities excluded from the solvent present in the hydrophobic core of proteins. HHP allows the solvation of buried amino acid side chains, thus shifting the equilibrium towards states of the studied molecule or molecular ensemble that occupy smaller volumes. HHP has long been used to dissociate multimeric proteins and protein aggregates and allows investigation of intermediate folding states, some of which are formed by proteins involved in human degenerative diseases, such as spongiform encephalopathies and Parkinson's disease, as well as cancer. When coupled with nuclear magnetic resonance and spectroscopic methods such as infrared and fluorescence spectroscopy, HHP treatment facilitates the understanding of protein folding and misfolding processes; the latter is related to protein aggregation into amyloid or amorphous species. In this review, we will address how HHP provides information about intermediate folding states and the aggregation processes of p53, which is related to cancer, and prion proteins, transthyretin and α-synuclein, which are related to human degenerative diseases.

Monoacylglycerols are increasingly used in several industrial applications as effective and cheap emulsifiers. In the present work monostearin synthesis has been studied, using lipase as a biocatalyst of the esterification reaction of stearic acid with (R,S)-1,2-O-iso-propylidene glycerol (solketal). The lipase from Candida antarctica (CaL B) was immobilized in AOT/isooctane water in oil microemulsions. Optimization of the reaction conditions have shown that the highest production (80% in 30 min) could be achieved at 40 °C, in microemulsions with relatively low water content (wo = 8). Kinetic studies have shown that the esterification reaction of stearic acid with solketal catalyzed by CaL B occurs via the ordered bi–bi mechanism, in which inhibition by the acid was identified. Moreover, at high fixed solketal concentrations a negative cooperativity is pronounced, which means that binding of the alcohol lowers the affinity of the enzyme for binding of the acid. Values of all kinetic parameters have been determined.

Inactivation of the tumor suppressor protein p53 by mutagenesis, chemical modification, protein-protein interaction, or aggregation has been associated with different human cancers. Although DNA is the typical substrate of p53, numerous studies have reported p53 interactions with RNA. Here, we have examined the effects of RNA of varied sequence, length, and origin on the mechanism of aggregation of the core domain of p53 (p53C) using light scattering, intrinsic fluorescence, transmission electron microscopy, thioflavin-T binding, seeding, and immunoblot assays. Our results are the first to demonstrate that RNA can modulate the aggregation of p53C and full-length p53. We found bimodal behavior of RNA in p53C aggregation. A low RNA:protein ratio (∼1:50) facilitates the accumulation of large amorphous aggregates of p53C. By contrast, at a high RNA:protein ratio (≥1:8), the amorphous aggregation of p53C is clearly suppressed. Instead, amyloid p53C oligomers are formed that can act as seeds nucleating de novo aggregation of p53C. We propose that structured RNAs prevent p53C aggregation through surface interaction and play a significant role in the regulation of the tumor suppressor protein.