RESUMO
BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.
Assuntos
Encéfalo , Síndrome de Creutzfeldt-Jakob , Cabras , Camundongos Transgênicos , Príons , Scrapie , Zoonoses , Animais , Síndrome de Creutzfeldt-Jakob/transmissão , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Scrapie/transmissão , Scrapie/patologia , Camundongos , Zoonoses/transmissão , Encéfalo/patologia , Encéfalo/metabolismo , Ovinos , Bovinos , Príons/metabolismo , Fenótipo , Baço/patologia , Encefalopatia Espongiforme Bovina/transmissão , Encefalopatia Espongiforme Bovina/patologia , Encefalopatia Espongiforme Bovina/metabolismo , Doenças das Cabras/transmissão , Doenças das Cabras/patologia , Modelos Animais de DoençasRESUMO
Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. The assembly of IBs is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We showed that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1-P interaction, thereby affecting the relative mobility of the proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules.
Assuntos
RNA , Vírus Sincicial Respiratório Humano , Alcaloides de Veratrum , Corpos de InclusãoRESUMO
Mammalian prions, the pathogens that cause transmissible spongiform encephalopathies, propagate by self-perpetuating the structural information stored in the abnormally folded, aggregated conformer (PrPSc) of the host-encoded prion protein (PrPC). To date, no structural model related to prion assembly organization satisfactorily describes how strain-specified structural information is encoded and by which mechanism this information is transferred to PrPC. To achieve progress on this issue, we correlated the PrPSc quaternary structural transition from three distinct prion strains during unfolding and refolding with their templating activity. We reveal the existence of a mesoscopic organization in PrPSc through the packing of a highly stable oligomeric elementary subunit (suPrP), in which the strain structural determinant (SSD) is encoded. Once kinetically trapped, this elementary subunit reversibly loses all replicative information. We demonstrate that acquisition of the templating interface and infectivity requires structural rearrangement of suPrP, in concert with its condensation. The existence of such an elementary brick scales down the SSD support to a small oligomer and provide a basis of reflexion for prion templating process and propagation.
Assuntos
Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Desdobramento de Proteína , Animais , Doenças Transmissíveis , Camundongos , Conformação ProteicaRESUMO
Compelling evidence supports a tight link between oxidative stress and protein aggregation processes, which are noticeably involved in the development of proteinopathies, such as Alzheimer's disease, Parkinson's disease, and prion disease. The literature is tremendously rich in studies that establish a functional link between both processes, revealing that oxidative stress can be either causative, or consecutive, to protein aggregation. Because oxidative stress monitoring is highly challenging and may often lead to artefactual results, cutting-edge technical tools have been developed recently in the redox field, improving the ability to measure oxidative perturbations in biological systems. This review aims at providing an update of the previously known functional links between oxidative stress and protein aggregation, thereby revisiting the long-established relationship between both processes.
Assuntos
Estresse Oxidativo , Agregação Patológica de Proteínas/metabolismo , Proteínas/metabolismo , Doença de Alzheimer/metabolismo , Animais , Humanos , Doença de Parkinson/metabolismo , Doenças Priônicas/metabolismo , Agregados ProteicosRESUMO
UNLABELLED: Prion diseases are characterized by conformational changes of a cellular prion protein (PrP(C)) into a ß-sheet-enriched and aggregated conformer (PrP(Sc)). Shadoo (Sho), a member of the prion protein family, is expressed in the central nervous system (CNS) and is highly conserved among vertebrates. On the basis of histoanatomical colocalization and sequence similarities, it is suspected that Sho and PrP may be functionally related. The downregulation of Sho expression during prion pathology and the direct interaction between Sho and PrP, as revealed by two-hybrid analysis, suggest a relationship between Sho and prion replication. Using biochemical and biophysical approaches, we demonstrate that Sho forms a 1:1 complex with full-length PrP with a dissociation constant in the micromolar range, and this interaction consequently modifies the PrP-folding pathway. Using a truncated PrP that mimics the C-terminal C1 fragment, an allosteric binding behavior with a Hill number of 4 was observed, suggesting that at least a tetramerization state occurs. A cell-based prion titration assay performed with different concentrations of Sho revealed an increase in the PrP(Sc) conversion rate in the presence of Sho. Collectively, our observations suggest that Sho can affect the prion replication process by (i) acting as a holdase and (ii) interfering with the dominant-negative inhibitor effect of the C1 fragment. IMPORTANCE: Since the inception of the prion theory, the search for a cofactor involved in the conversion process has been an active field of research. Although the PrP interactome presents a broad landscape, candidates corresponding to specific criteria for cofactors are currently missing. Here, we describe for the first time that Sho can affect PrP structural dynamics and therefore increase the prion conversion rate. A biochemical characterization of Sho-PrP indicates that Sho acts as an ATP-independent holdase.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Príons/metabolismo , Dobramento de Proteína , Animais , Proteínas Ligadas por GPI , Camundongos , Ligação Proteica , Multimerização Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
During rotavirus infection, replication and packaging of the viral genome occur in viral factories, termed viroplasms. The viral nonstructural protein NSP5 is a major building block of viroplasms; it recruits the viral polymerase VP1, the core protein VP2, and the ATPase NSP2 inside the viroplasm to form the viral replication complex. Here we report that NSP5 is a unique viral metalloprotein that coordinates a [2Fe-2S] iron-sulfur cluster as demonstrated by the metal and labile sulfide contents, UV-visible light absorption, and electron paramagnetic resonance. Point mutations in NSP5 allowed us to identify C171 and C174, arranged in a CXC motif, as essential residues for cluster coordination. When coexpressed with NSP2, an NSP5 mutant devoid of the iron-sulfur cluster still forms viroplasm-like structures. The cluster is therefore neither involved in the interaction with NSP2 nor in the formation of viroplasm-like structures and thus presumably in viroplasm formation. Finally, we show using microscale thermophoresis that the iron-sulfur cluster modulates the affinity of NSP5 for single-stranded RNA. Because the cluster is near the binding sites of both the polymerase VP1 and the ATPase NSP2, we anticipate that this cluster is crucial for NSP5 functions, in either packaging or replication of the viral genome.
Assuntos
Metaloproteínas/química , RNA Viral/química , Rotavirus/química , Proteínas não Estruturais Virais/química , Humanos , Ferro/química , Ferro/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Mutação Puntual , RNA Viral/genética , RNA Viral/metabolismo , Rotavirus/fisiologia , Infecções por Rotavirus/genética , Infecções por Rotavirus/metabolismo , Espectrofotometria Ultravioleta , Enxofre/química , Enxofre/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus/fisiologia , Replicação Viral/fisiologiaRESUMO
Microtubules, components of the cell cytoskeleton, play a central role in cellular trafficking. Here we show that rotavirus infection leads to a remodeling of the microtubule network together with the formation of tubulin granules. While most microtubules surrounding the nucleus depolymerize, others appear packed at the cell periphery. In microtubule depolymerization areas, tubulin granules are observed; they colocalize with viroplasms, viral compartments formed by interactions between rotavirus proteins NSP2 and NSP5. With purified proteins, we show that tubulin directly interacts in vitro with NSP2 but not with NSP5. The binding of NSP2 to tubulin is independent of its phosphatase activity. The comparison of three-dimensional (3-D) reconstructions of NSP2 octamers alone or associated with tubulin reveals electron densities in the positively charged grooves of NSP2 that we attribute to tubulin. Site-directed mutagenesis of NSP2 and competition assays between RNA and tubulin for NSP2 binding confirm that tubulin binds to these charged grooves of NSP2. Although the tubulin position within NSP2 grooves cannot be precisely determined, the tubulin C-terminal H12 alpha-helix could be involved in the interaction. NSP2 overexpression and rotavirus infection produce similar effects on the microtubule network. NSP2 depolymerizes microtubules and leads to tubulin granule formation. Our results demonstrate that tubulin is a viroplasm component and reveal an original mechanism. Tubulin sequestration by NSP2 induces microtubule depolymerization. This depolymerization probably reroutes the cell machinery by inhibiting trafficking and functions potentially involved in defenses to viral infections.
Assuntos
Microtúbulos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Infecções por Rotavirus/metabolismo , Rotavirus/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nocodazol/farmacologia , Paclitaxel/farmacologia , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/ultraestrutura , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestrutura , Moduladores de Tubulina/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/ultraestruturaRESUMO
Prions are neurotropic pathogens composed of misfolded assemblies of the host-encoded prion protein PrPC which replicate by recruitment and conversion of further PrPC by an autocatalytic seeding polymerization process. While it has long been shown that mouse-adapted prions cannot replicate and are rapidly cleared in transgenic PrP0/0 mice invalidated for PrPC, these experiments have not been done with other prions, including from natural resources, and more sensitive methods to detect prion biological activity. Using transgenic mice expressing human PrP to bioassay prion infectivity and RT-QuIC cell-free assay to measure prion seeding activity, we report that prions responsible for the most prevalent form of sporadic Creutzfeldt-Jakob disease in human (MM1-sCJD) can persist indefinitely in the brain of intra-cerebrally inoculated PrP0/0 mice. While low levels of seeding activity were measured by RT-QuIC in the brain of the challenged PrP0/0 mice, the bio-indicator humanized mice succumbed at a high attack rate, suggesting relatively high levels of persistent infectivity. Remarkably, these humanized mice succumbed with delayed kinetics as compared to MM1-sCJD prions directly inoculated at low doses, including the limiting one. Yet, the disease that did occur in the humanized mice on primary and subsequent back-passage from PrP0/0 mice shared the neuropathological and molecular characteristics of MM1-sCJD prions, suggesting no apparent strain evolution during lifelong dormancy in PrP0/0 brain. Thus, MM1-sCJD prions can persist for the entire life in PrP0/0 brain with potential disease potentiation on retrotransmission to susceptible hosts. These findings highlight the capacity of prions to persist and rejuvenate in non-replicative environments, interrogate on the type of prion assemblies at work and alert on the risk of indefinite prion persistence with PrP-lowering therapeutic strategies.
RESUMO
Genetically encoded ratiometric fluorescent probes are cutting-edge tools in biology. They allow precise and dynamic measurement of various physiological parameters within cell compartments. Because data extraction and analysis are time consuming and may lead to inconsistencies between results, we describe here a standardized pipeline forâ¢Semi-automated treatment of time-lapse fluorescence microscopy images.â¢Quantification of individual cell signal.â¢Statistical analysis of the data.First, a dedicated macro was developed using the FIJI software to reproducibly quantify the fluorescence ratio as a function of time. Raw data are then exported and analyzed using R and MATLAB softwares. Calculation and statistical analysis of selected graphic parameters are performed. In addition, a functional principal component analysis allows summarizing the dataset. Finally, a principal component analysis is performed to check consistency and final analysis is presented as a visual diagram. The method is adapted to any ratiometric fluorescent probe. As an example, the analysis of the cytoplasmic HyPer probe in response to an acute cell treatment with increasing amounts of hydrogen peroxide is shown. In conclusion, the pipeline allows to save time and analyze a larger amount of samples while reducing manual interventions and consequently increasing the robustness of the analysis.
RESUMO
The abnormal protein aggregates in progressive neurodegenerative disorders, such as Alzheimer's, Parkinson's and prion diseases, adopt a generic structural form called amyloid fibrils. The precise amyloid fold can differ between patients and these differences are related to distinct neuropathological phenotypes of the diseases. A key focus in current research is the molecular mechanism governing such structural diversity, known as amyloid polymorphism. In this review, we focus on our recent work on recombinant prion protein (recPrP) and the use of pressure as a variable for perturbing protein structure. We suggest that the amyloid polymorphism is based on volumetric features. Accordingly, pressure is the thermodynamic parameter that fits best to exploit volume differences within the states of a chemical reaction, since it shifts the equilibrium constant to the state that has the smaller volume. In this context, there are analogies with the process of correct protein folding, the high pressure-induced effects of which have been studied for more than a century and which provides a valuable source of inspiration. We present a short overview of this background and review our recent results regarding the folding, misfolding, and aggregation-disaggregation of recPrP under pressure. We present preliminary experiments aimed at identifying how prion protein fibril diversity is related to the quaternary structure by using pressure and varying protein sequences. Finally, we consider outstanding questions and testable mechanistic hypotheses regarding the multiplicity of states in the amyloid fold.
Assuntos
Amiloide/genética , Polimorfismo Genético , Pressão , Proteínas Priônicas/genética , Amiloide/química , Humanos , Doenças Priônicas , Príons/química , Agregados Proteicos , Conformação Proteica , Dobramento de Proteína , TermodinâmicaRESUMO
The prion protein (PrP) misfolds and assembles into a wide spectrum of self-propagating quaternary structures, designated PrPSc. These various PrP superstructures can be functionally different, conferring clinically distinctive symptomatology, neuropathology and infectious character to the associated prion diseases. However, a satisfying molecular basis of PrP structural diversity is lacking in the literature. To provide mechanistic insights into the etiology of PrP polymorphism, we have engineered a set of 6 variants of the human protein and obtained PrP amyloid fibrils. We show that pressure induces dissociation of the fibrils, albeit with different kinetics. In addition, by focusing on the generic properties of amyloid fibrils, such as the thioflavin T binding capacities and the PK-resistance, we reveal an unprecedented structure-barostability phenomenological relationship. We propose that the structural diversity of PrP fibrils encompass a multiplicity of packing defects (water-excluded cavities) in their hydrophobic cores, and that the resultant sensitivity to pressure should be considered as a general molecular criterion to accurately define fibril morphotypes. We anticipate that our insights into sequence-dependent fibrillation and conformational stability will shed light on the highly-nuanced prion strain phenomenon and open the opportunity to explain different PrP conformations in terms of volumetric physics.
Assuntos
Pressão , Proteínas Priônicas/química , Agregados Proteicos , Substituição de Aminoácidos , Amiloide/química , Benzotiazóis/metabolismo , Humanos , Modelos Moleculares , Proteínas Priônicas/genética , Agregados Proteicos/genética , Conformação Proteica , Engenharia de ProteínasRESUMO
In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.
Assuntos
Células Dendríticas Foliculares/metabolismo , Granuloma/patologia , Doenças Priônicas/patologia , Proteínas Priônicas/metabolismo , Animais , Antígenos de Superfície/metabolismo , Modelos Animais de Doenças , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Leite/metabolismo , Proteínas Priônicas/genética , Proteínas Priônicas/isolamento & purificação , Proteínas Priônicas/toxicidade , Dobramento de Proteína , Ovinos , Baço/citologia , TropismoRESUMO
In mammals, Prion pathology refers to a class of infectious neuropathologies whose mechanism is based on the self-perpetuation of structural information stored in the pathological conformer. The characterisation of the PrP folding landscape has revealed the existence of a plethora of pathways conducing to the formation of structurally different assemblies with different biological properties. However, the biochemical interconnection between these diverse assemblies remains unclear. The PrP oligomerisation process leads to the formation of neurotoxic and soluble assemblies called O1 oligomers with a high size heterodispersity. By combining the measurements in time of size distribution and average size with kinetic models and data assimilation, we revealed the existence of at least two structurally distinct sets of assemblies, termed Oa and Ob, forming O1 assemblies. We propose a kinetic model representing the main processes in prion aggregation pathway: polymerisation, depolymerisation, and disintegration. The two groups interact by exchanging monomers through a disintegration process that increases the size of Oa. Our observations suggest that PrP oligomers constitute a highly dynamic population.
Assuntos
Príons/química , Agregados Proteicos , Agregação Patológica de Proteínas , Multimerização Proteica , Algoritmos , Animais , Simulação por Computador , Cinética , Modelos Químicos , Desdobramento de Proteína , OvinosRESUMO
DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((253)KKRK(256)) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Dicroísmo Circular , Clonagem Molecular , DNA/química , Drosophila melanogaster , Eletroforese em Gel de Poliacrilamida , Lisina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Triptofano/químicaRESUMO
The protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1: a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteína HMGB1/genética , Proteínas de Grupo de Alta Mobilidade/genética , Animais , Cromatina/genética , Dicroísmo Circular , DNA/química , DNA/metabolismo , DNA Ligases/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Glutamina/química , Glutamina/genética , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Proteína HMGB2/química , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Plasmídeos , Ratos , Proteínas Recombinantes/genética , Transcrição GênicaRESUMO
Abnormal accumulation of alpha-synuclein filaments in Lewy bodies is a neuropathological hallmark of Parkinson's disease and sequestration of cellular protein into these protein aggregates may contribute to the degenerative process. We identified the transcriptional co-factor high mobility group protein 1 (HMGB-1) as a ligand for alpha-synuclein filaments by a filament spin-down technique, mass spectrometric peptide mapping and immunoblotting. HMGB-1 binds preferentially to aggregated alpha-synuclein and is present in alpha-synuclein filament-containing Lewy bodies isolated from brain tissue affected with dementia with Lewy bodies or Parkinson's disease. Our results demonstrate that alpha-synuclein filaments hold the potential for disturbing the cellular gene expression as they can sequester a component involved in cellular transcription regulation.
Assuntos
Proteína HMGB1/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteína HMGB1/análise , Proteína HMGB1/química , Proteína HMGB1/ultraestrutura , Humanos , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Isótopos de Iodo/metabolismo , Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/metabolismo , Ligantes , Microscopia Imunoeletrônica/métodos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/ultraestrutura , Neuritos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Mapeamento de Peptídeos/métodos , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Sinucleínas , alfa-SinucleínaRESUMO
Rotavirus is one of the leading agents of gastroenteritis worldwide. During infection, viral factories (viroplasms) are formed. The rotavirus nonstructural proteins NSP5 and NSP2 are the major building blocks of viroplasms; however, NSP5 function and organisation remain elusive. In this report, we present a structural characterisation of NSP5. Multi-angle laser light scattering, sedimentation velocity and equilibrium sedimentation experiments demonstrate that recombinant full-length NSP5 forms a decamer in solution. Far-Western, pull-down and multi-angle laser light scattering experiments show that NSP5 has two oligomerisation regions. The first region, residues 103-146, is involved in NSP5 dimerisation, whereas the second region, residues 189-198, is responsible for NSP5 decamerisation. Circular dichroism analyses of full-length and truncated forms of NSP5 reveal that the decamerisation region is helical, whereas the dimerisation region involves ß-sheets. From these circular dichroism experiments, we also show that the NSP5 protomers contain two α-helices, a disordered N-terminal half and a C-terminal half that is primarily composed of ß-sheet folds. This extensive structural characterisation of NSP5 led us to propose a model for its quaternary organisation. Finally, co-expression of NSP5 fragments and NSP2 in uninfected cells shows that the NSP5 decamerisation region is required for viroplasm-like structure formation. However, in vitro, the NSP5 decamerisation region partially inhibits the NSP2-NSP5 interaction. Our NSP5 model suggests that steric hindrance prevents NSP2 from binding to all NSP5 protomers. Some protomers may thus be free to interact with other NSP5 binding partners, such as viral RNAs and the viral polymerase VP1, to perform functions other than viroplasm organisation.
Assuntos
Proteínas não Estruturais Virais/química , Far-Western Blotting , Dicroísmo Circular , Modelos Moleculares , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise Espectral Raman , Ultracentrifugação , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form. PRINCIPAL FINDINGS: Here we report that this free form of Brk1 is composed of homotrimers. Using a novel assay in which purified free Brk1 is electroporated into HeLa cells, we were able to follow its biochemical fate in cells and to show that free Brk1 becomes incorporated into the Wave complex. Importantly, incorporation of free Brk1 into the Wave complex was blocked upon inhibition of protein synthesis and incorporated Brk1 was found to associate preferentially with neosynthesized subunits. Brk1 depleted HeLa cells were found to bleb, as were Nap1, Wave2 or ARPC2 depleted cells, suggesting that this blebbing phenotype of Brk1 depleted cells is due to an impairment of the Wave complex function rather than a specific function of free Brk1. Blebs of Brk1 depleted cells were emitted at sites where lamellipodia and membrane ruffles were normally emitted. In Brk1 depleted cells, the electroporation of free Brk1 was sufficient to restore Wave complex assembly and to rescue the blebbing phenotype. CONCLUSION: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of a functional Wave complex.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Sequência de Aminoácidos , Biopolímeros , Linhagem Celular , Proteínas do Citoesqueleto/química , Eletroforese em Gel de Poliacrilamida , Eletroporação , Humanos , Imunoprecipitação , Dados de Sequência Molecular , TransfecçãoRESUMO
Myosin VI contains an inserted sequence that is unique among myosin superfamily members and has been suggested to be a determinant of the reverse directionality and unusual motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds one calmodulin (CaM) by means of a single "IQ motif". Using truncations of the myosin VI protein and electrospray ionization(ESI)-MS, we demonstrate that in fact each myosin VI head binds two CaMs. One CaM binds to a conventional IQ motif either with or without calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The second CaM binds to a unique insertion between the converter region and IQ motif. This unusual CaM-binding site normally binds CaM with four Ca2+ and can bind only if the C-terminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide contribute to the Ca(2+)-CaM binding, as truncations that eliminate elements of the MD alter CaM binding and allow calcium dissociation. We suggest that the Ca(2+)-CaM bound to the unique insert represents a structural CaM, and not a calcium sensor or regulatory component of the motor. This structure is likely an integral part of the myosin VI "converter" region and repositions the myosin VI "lever arm" to allow reverse direction (minus-end) motility on actin.