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The Nipah and Hendra viruses are severe human pathogens. In addition to the P protein, their P gene also encodes the V and W proteins that share with P their N-terminal intrinsically disordered domain (NTD) and possess distinct C-terminal domains (CTDs). The W protein is a key player in the evasion of the host innate immune response. We previously showed that the W proteins are intrinsically disordered and can form amyloid-like fibrils. However, structural information on W CTD (CTDW) and its potential contribution to the fibrillation process is lacking. In this study, we demonstrate that CTDWS are disordered and able to form dimers mediated by disulfide bridges. We also show that the NTD and the CTDW interact with each other and that this interaction triggers both a gain of secondary structure and a chain compaction within the NTD. Finally, despite the lack of intrinsic fibrillogenic properties, we show that the CTDW favors the formation of fibrils by the NTD both in cis and in trans. Altogether, the results herein presented shed light on the molecular mechanisms underlying Henipavirus pathogenesis and may thus contribute to the development of targeted therapies.
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The RNA genome of measles virus is encapsidated by the nucleoprotein within a helical nucleocapsid that serves as template for both transcription and replication. The intrinsically disordered domain of the nucleoprotein (NTAIL), partly protruding outward from the nucleocapsid, is essential for binding the polymerase complex responsible for viral transcription and replication. As for many IDPs, binding of NTAIL occurs through a short molecular recognition element (MoRE) that folds upon binding, with the majority of NTAIL remaining disordered. Though NTAIL regions far from the MoRE influence the binding affinity, interactions between them and the MoRE have not been investigated in depth. Using an integrated approach, relying on photo-induced electron transfer (PET) experiments between tryptophan and cysteine pairs placed at different positions in the protein under varying salt and pH conditions, combined with simulations and analytical models, we identified transient interactions between two disordered regions distant in sequence, which dominate NTAIL dynamics, and regulate the conformational preferences of both the MoRE and the entire NTAIL domain. Co-evolutionary analysis corroborates our findings, and suggests an important functional role for the same intramolecular interactions. We propose mechanisms by which these non-local interactions may regulate binding to the phosphoprotein, polymerase recruitment, and ultimately viral transcription and replication. Our findings may be extended to other IDPs, where non-local intra-protein interactions affect the conformational preferences of intermolecular binding sites.
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In the field of virology, liquid-liquid phase separation (LLPS) has emerged as a pivotal mechanism enabling the compartmentalization required for specific steps of the viral replication cycle [...].
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Virosis , Humanos , Virosis/virología , Replicación Viral , Extracción Líquido-Líquido/métodos , Separación de FasesRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.
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Proteínas Fúngicas , Polisacáridos , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Difracción de Rayos X , Polisacáridos/metabolismo , Celulosa/metabolismoRESUMEN
Liquid-liquid phase separation (LLPS) is pivotal in forming biomolecular condensates, which are crucial in several biological processes. Intrinsically disordered regions (IDRs) are typically responsible for driving LLPS due to their multivalency and high content of charged residues that enable the establishment of electrostatic interactions. In our study, we examined the role of charge distribution in the condensation of the disordered N-terminal domain of human topoisomerase I (hNTD). hNTD is densely charged with oppositely charged residues evenly distributed along the sequence. Its LLPS behavior was compared with that of charge permutants exhibiting varying degrees of charge segregation. At low salt concentrations, hNTD undergoes LLPS. However, LLPS is inhibited by high concentrations of salt and RNA, disrupting electrostatic interactions. Our findings show that, in hNTD, moderate charge segregation promotes the formation of liquid condensates that are sensitive to salt and RNA, whereas marked charge segregation results in the formation of aberrant condensates. Although our study is based on a limited set of protein variants, it supports the applicability of the "stickers-and-spacers" model to biomolecular condensates involving highly charged IDRs. These results may help generate reliable models of the overall LLPS behavior of supercharged polypeptides.
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ADN-Topoisomerasas de Tipo I , ARN , Humanos , ADN-Topoisomerasas de Tipo I/genética , Electricidad EstáticaRESUMEN
Amyloidoses are an array of diseases associated with the aggregation of proteins into fibrils. While it was previously thought that amyloid fibril-forming proteins are exclusively host-cell encoded, recent studies have revealed that pathogenic viruses can form amyloid-like fibrils too. Intriguingly, viral amyloids are often composed of virulence factors, known for their contribution to cell death and disease progression. In this review, we survey the literature about viral proteins capable of forming amyloid-like fibrils. The molecular and cellular mechanisms underlying the formation of viral amyloid-like aggregates are explored. In addition, we discuss the functional implications for viral amplification and the complex interplay between viral amyloids, biological functions, virulence, and virus-induced pathologies.
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Amiloide , Proteínas Amiloidogénicas , Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Factores de Virulencia , AntiviralesRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and ß-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle.IMPORTANCELytic polysaccharide monooxygenases (LPMOs) have been mainly studied in a biotechnological context for the efficient degradation of recalcitrant polysaccharides. Only recently, alternative roles and paradigms begin to emerge. In this study, we provide evidence that the AA10 LPMO from the phytopathogen Ustilago maydis is active against fungal cell wall chitin. Given that chitin-active LPMOs are commonly found in microbes, it is important to consider fungal cell wall as a potential target for this enigmatic class of enzymes.
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Quitina , Polisacáridos , Quitina/metabolismo , Polisacáridos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Celulosa/metabolismo , Pared Celular/metabolismoRESUMEN
An unambiguous description of an experiment, and the subsequent biological observation, is vital for accurate data interpretation. Minimum information guidelines define the fundamental complement of data that can support an unambiguous conclusion based on experimental observations. We present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the parameters required for the wider scientific community to understand the findings of an experiment studying the structural properties of intrinsically disordered regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardize the dissemination of the key metadata on an IDR experiment by IDR data sources.
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Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Conformación ProteicaRESUMEN
Until the late 1990s, we believed that protein function required a unique, well-defined 3D structure encrypted in the amino acid sequence [...].
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Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Pliegue de Proteína , Conformación Proteica , Modelos Moleculares , Secuencia de AminoácidosRESUMEN
The split-green fluorescent protein (GFP) reassembly assay is a well-established approach to study protein-protein interactions (PPIs). In this assay, when two interacting proteins X and Y, respectively fused to residues 1-157 and to residues 158-237 of GFP, are co-expressed in E. coli, the two GFP halves are brought to sufficient proximity to reassociate and fold to recreate the functional GFP. At constant protein expression level, the intensity of fluorescence produced by the bacteria is proportional to the binding affinity of X to Y. We hypothesized that adding a third partner (Z) endowed with an affinity for either X or Y would lead to an in vivo competition assay. We report here the different steps of the set-up of this competition assay, and define the experimental conditions required to obtained reliable results. Results show that this competition assay is a potentially interesting tool for screening libraries of binding inhibitors, Z being either a protein or a chemical reagent.
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Escherichia coli , Proteínas Fluorescentes Verdes/metabolismo , Unión Proteica , Escherichia coli/metabolismo , FluorescenciaRESUMEN
Liquid-liquid phase separation (LLPS) is responsible for the formation of so-called membrane-less organelles (MLOs) that are essential for the spatio-temporal organization of the cell. Intrinsically disordered proteins (IDPs) or regions (IDRs), either alone or in conjunction with nucleic acids, are involved in the formation of these intracellular condensates. Notably, viruses exploit LLPS at their own benefit to form viral replication compartments. Beyond giving rise to biomolecular condensates, viral proteins are also known to partition into cellular MLOs, thus raising the question as to whether these cellular phase-separating proteins are drivers of LLPS or behave as clients/regulators. Here, we focus on a set of eukaryotic proteins that are either sequestered in viral factories or colocalize with viral proteins within cellular MLOs, with the primary goal of gathering organized, predicted, and experimental information on these proteins, which constitute promising targets for innovative antiviral strategies. Using various computational approaches, we thoroughly investigated their disorder content and inherent propensity to undergo LLPS, along with their biological functions and interactivity networks. Results show that these proteins are on average, though to varying degrees, enriched in disorder, with their propensity for phase separation being correlated, as expected, with their disorder content. A trend, which awaits further validation, tends to emerge whereby the most disordered proteins serve as drivers, while more ordered cellular proteins tend instead to be clients of viral factories. In light of their high disorder content and their annotated LLPS behavior, most proteins in our data set are drivers or co-drivers of molecular condensation, foreshadowing a key role of these cellular proteins in the scaffolding of viral infection-related MLOs.
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Proteínas Intrínsecamente Desordenadas , Virosis , Humanos , Orgánulos/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Virales/metabolismo , Virosis/metabolismo , Eucariontes/metabolismoRESUMEN
The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein-protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four ß-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan's lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results.
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Bioensayo , Escherichia coli , Proteínas Fluorescentes Verdes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Measles, Nipah and Hendra viruses are severe human pathogens within the Paramyxoviridae family. Their non-segmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the viral RNA-dependent-RNA-polymerase (RpRd) for transcription and replication. The RpRd is a complex made of the large protein (L) and of the phosphoprotein (P), the latter serving as an obligate polymerase cofactor and as a chaperon for N. Both the N and P proteins are enriched in intrinsically disordered regions (IDRs), i.e. regions devoid of stable secondary and tertiary structure. N possesses a C-terminal IDR (NTAIL), while P consists of a large, intrinsically disordered N-terminal domain (NTD) and a C-terminal domain (CTD) encompassing alternating disordered and ordered regions. The V and W proteins, two non-structural proteins that are encoded by the P gene via a mechanism of co-transcriptional edition of the P mRNA, are prevalently disordered too, sharing with P the disordered NTD. They are key players in the evasion of the host antiviral response and were shown to phase separate and to form amyloid-like fibrils in vitro. In this review, we summarize the available information on IDRs within the N, P, V and W proteins from these three model paramyxoviruses and describe their molecular partnership. We discuss the functional benefit of disorder to virus replication in light of the critical role of IDRs in affording promiscuity, multifunctionality, fine regulation of interaction strength, scaffolding functions and in promoting liquid-liquid phase separation and fibrillation.
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Virus Hendra , Virus del Sarampión , Virus Nipah , Replicación Viral , Virus Hendra/genética , Virus Hendra/fisiología , Nucleoproteínas/química , Nucleoproteínas/genética , ARN , Virus del Sarampión/genética , Virus del Sarampión/fisiología , Virus Nipah/genética , Virus Nipah/fisiologíaRESUMEN
Background: Patients with multiple sclerosis (MS) demonstrate higher seroprevalence of Epstein-Barr virus (EBV) and increased anti-EBV IgG levels in serum compared with healthy controls. Intrathecal antibody production to measles virus (MeV) is a common finding in patients with MS. Objective: To measure serum IgG reactivity to EBV glycoprotein 350 (gp350) and MeV nucleocapsid protein (NCORE) in patients with MS and healthy controls and to determine if reactivity changed in patients during interferon beta (IFNß) and/or natalizumab (NAT) treatment. A secondary aim was to determine the seroprevalence of EBV in patients and controls. Methods: Patients with MS (n=728) were included from the Swedish pharmacovigilance study for NAT. Paired serum samples from 714 patients drawn before and during NAT treatment and paired samples from 170 patients during prior IFNß treatment were analysed. In total, 156 patients were included in both groups. Samples from 144 matched blood donors served as controls. Indirect ELISA was applied using recombinant EBVgp350 and MeV NCORE as antigens. EBVgp350 IgG seronegative samples were also analysed using EBV nuclear antigen 1 and viral capsid antigen (VCA). Results: Patients with MS showed higher serum levels of anti-EBVgp350 and anti-MeV NCORE IgG compared with controls. During NAT treatment, the levels of anti-EBVgp350 and anti-MeV NCORE IgG declined, compared with the relatively stable levels noted during prior IFNß treatment. Ten patients failed to demonstrate anti-EBVgp350 IgG but did show detectable anti-VCA IgG, indicating EBV seropositivity. In contrast, 10/144 controls were EBV seronegative. Conclusions: Treatment with NAT, which is considered a selective immunosuppressive agent with a compartmentalised effect on the central nervous system, appeared to be associated with a moderate decrease in circulating IgG levels to EBVgp350 and MeV NCORE. All patients with MS were EBV IgG seropositive, supporting the potential role of EBV in the pathogenesis of MS.
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In the last two decades it has become increasingly evident that a large number of proteins adopt either a fully or a partially disordered conformation. Intrinsically disordered proteins are ubiquitous proteins that fulfill essential biological functions while lacking a stable 3D structure. Their conformational heterogeneity is encoded by the amino acid sequence, thereby allowing intrinsically disordered proteins or regions to be recognized based on their sequence properties. The identification of disordered regions facilitates the functional annotation of proteins and is instrumental for delineating boundaries of protein domains amenable to crystallization. This chapter focuses on the methods currently employed for predicting protein disorder and identifying intrinsically disordered binding sites.
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Proteínas Intrínsecamente Desordenadas , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Intrínsecamente Desordenadas/química , Unión Proteica , Conformación Proteica , Dominios ProteicosRESUMEN
Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties.
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Proteínas Intrínsecamente Desordenadas , Aminoácidos/química , Proteínas Intrínsecamente Desordenadas/química , Prolina , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
Henipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V and W proteins which share with P their N-terminal, intrinsically disordered domain (NTD) and possess a unique C-terminal domain. Henipavirus W proteins antagonize interferon (IFN) signaling through NTD-mediated binding to STAT1 and STAT4, and prevent type I IFN expression and production of chemokines. Structural and molecular information on Henipavirus W proteins is lacking. By combining various bioinformatic approaches, we herein show that the Henipaviruses W proteins are predicted to be prevalently disordered and yet to contain short order-prone segments. Using limited proteolysis, differential scanning fluorimetry, analytical size exclusion chromatography, far-UV circular dichroism and small-angle X-ray scattering, we experimentally confirmed their overall disordered nature. In addition, using Congo red and Thioflavin T binding assays and negative-staining transmission electron microscopy, we show that the W proteins phase separate to form amyloid-like fibrils. The present study provides an additional example, among the few reported so far, of a viral protein forming amyloid-like fibrils, therefore significantly contributing to enlarge our currently limited knowledge of viral amyloids. In light of the critical role of the Henipavirus W proteins in evading the host innate immune response and of the functional role of phase separation in biology, these studies provide a conceptual asset to further investigate the functional impact of the phase separation abilities of the W proteins.
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Amiloide/metabolismo , Henipavirus/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Benzotiazoles/metabolismo , Dicroismo Circular , Simulación por Computador , Rojo Congo/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Moleculares , Dominios Proteicos , Proteolisis , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.
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Virus Hendra , Infecciones por Henipavirus , Virus Nipah , Humanos , Virus Hendra/genética , Interferones/metabolismo , Inmunidad InnataRESUMEN
The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.
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Bases de Datos de Proteínas , Proteínas Intrínsecamente Desordenadas/metabolismo , Anotación de Secuencia Molecular , Programas Informáticos , Secuencia de Aminoácidos , ADN/genética , ADN/metabolismo , Conjuntos de Datos como Asunto , Ontología de Genes , Humanos , Internet , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Unión Proteica , ARN/genética , ARN/metabolismoRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes secreted by many organisms and viruses. LPMOs catalyze the oxidative cleavage of different types of polysaccharides and are today divided into eight families (AA9-11, AA13-17) within the Auxiliary Activity enzyme class of the CAZy database. LPMOs minimal architecture encompasses a catalytic domain, to which can be appended a carbohydrate-binding module. Intriguingly, we observed that some LPMO sequences also display a C-terminal extension of varying length not associated with any known function or fold. Here, we analyzed 27,060 sequences from different LPMO families and show that 60% have a C-terminal extension predicted to be intrinsically disordered. Our analysis shows that these disordered C-terminal regions (dCTRs) are widespread in all LPMO families (except AA13) and differ in terms of sequence length and amino-acid composition. Noteworthily, these dCTRs have so far only been observed in LPMOs. LPMO-dCTRs share a common polyampholytic nature and an enrichment in serine and threonine residues, suggesting that they undergo post-translational modifications. Interestingly, dCTRs from AA11 and AA15 are enriched in redox-sensitive, conditionally disordered regions. The widespread occurrence of dCTRs in LPMOs from evolutionarily very divergent organisms, hints at a possible functional role and opens new prospects in the field of LPMOs.