RESUMEN
Biomolecular condensates (BMCs) exhibit physiological and pathological relevance in biological systems. Both liquid and solid condensates play significant roles in the spatiotemporal regulation and organization of macromolecules and their biological activities. Some pathological solid condensates, such as Lewy Bodies and other fibrillar aggregates, have been hypothesized to originate from liquid condensates. With the prevalence of BMCs having functional and dysfunctional roles, it is imperative to understand the mechanism of biomolecular condensate formation and initiation. Using the low-complexity domain (LCD) of heterogenous ribonuclear protein A1 (hnRNPA1) as our model, we monitored initial assembly events using dynamic light scattering (DLS) while modulating pH and salt conditions to perturb macromolecule and condensate properties. We observed the formation of nanometer-sized BMCs (nano-condensates) distinct from protein monomers and micron-sized condensates. We also observed that conditions that solubilize micron-sized protein condensates do not solubilize nano-condensates, indicating that the balance of forces that stabilize nano-condensates and micron-sized condensates are distinct. These findings provide insight into the forces that drive protein phase separation and potential nucleation structures of macromolecular condensation.
Asunto(s)
Dispersión Dinámica de Luz , Ribonucleoproteína Nuclear Heterogénea A1 , Humanos , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1/química , Dominios Proteicos , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , Concentración de Iones de HidrógenoRESUMEN
NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of cis PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and cis Pro enhancement at both PEST sites lead to a 5-10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The cis-populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.
Asunto(s)
Peptidilprolil Isomerasa de Interacción con NIMA , Proteína Homeótica Nanog , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Proteína Homeótica Nanog/metabolismo , Proteína Homeótica Nanog/genética , Fosforilación , Humanos , Estabilidad Proteica , Unión Proteica , EstereoisomerismoRESUMEN
Cellular deposition of protein aggregates, one of the hallmarks of neurodegeneration, disrupts cellular functions and leads to neuronal death. Mutations, posttranslational modifications, and truncations are common molecular underpinnings in the formation of aberrant protein conformations that seed aggregation. The major proteins involved in neurodegeneration include amyloid beta (Aß) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, and TAR DNA-binding protein (TDP-43) in amyotrophic lateral sclerosis (ALS). These proteins are described as intrinsically disordered and possess enhanced ability to partition into biomolecular condensates. In this review, we discuss the role of protein misfolding and aggregation in neurodegenerative diseases, specifically highlighting implications of changes to the primary/secondary (mutations, posttranslational modifications, and truncations) and the quaternary/supramolecular (oligomerization and condensation) structural landscapes for the four aforementioned proteins. Understanding these aggregation mechanisms provides insights into neurodegenerative diseases and their common underlying molecular pathology.
Asunto(s)
Enfermedad de Alzheimer , Esclerosis Amiotrófica Lateral , Proteínas Intrínsecamente Desordenadas , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Esclerosis Amiotrófica Lateral/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas tauRESUMEN
Biomolecular condensates of ribonucleoproteins (RNPs) such as the transactivation response element (TAR) DNA-binding protein 43 (TDP-43) arise from liquid-liquid phase separation (LLPS) and play vital roles in various biological processes including the formation-dissolution of stress granules (SGs). These condensates are thought to be directly linked to neurodegenerative diseases, providing a depot of aggregation-prone proteins and serving as a cauldron of protein aggregation and fibrillation. Despite recent research efforts, biochemical processes and rearrangements within biomolecular condensates that trigger subsequent protein misfolding and aggregation remain to be elucidated. Fluorescence lifetime imaging microscopy (FLIM) provides a minimally intrusive high-sensitivity and high-resolution imaging method to monitor in-droplet spatiotemporal changes that initiate and lead to protein aggregation. In this chapter, we describe a FLIM application for characterizing chemical chaperone-assisted decoupling of TDP-43 liquid-liquid phase separation and aggregation/fibrillation, highlighting potential therapeutic strategies to combat pathological RNP-associated aggregates without compromising cellular stress responses.
Asunto(s)
Condensados Biomoleculares , Agregado de Proteínas , Proteínas de Unión al ADN/metabolismo , Microscopía Fluorescente , Ribonucleoproteínas/metabolismoRESUMEN
Human NANOG expression resets stem cells to ground-state pluripotency. Here we identify the unique features of human NANOG that relate to its dose-sensitive function as a master transcription factor. NANOG is largely disordered, with a C-terminal prion-like domain that phase-transitions to gel-like condensates. Full-length NANOG readily forms higher-order oligomers at low nanomolar concentrations, orders of magnitude lower than typical amyloids. Using single-molecule Förster resonance energy transfer and fluorescence cross-correlation techniques, we show that NANOG oligomerization is essential for bridging DNA elements in vitro. Using chromatin immunoprecipitation sequencing and Hi-C 3.0 in cells, we validate that NANOG prion-like domain assembly is essential for specific DNA recognition and distant chromatin interactions. Our results provide a physical basis for the indispensable role of NANOG in shaping the pluripotent genome. NANOG's unique ability to form prion-like assemblies could provide a cooperative and concerted DNA bridging mechanism that is essential for chromatin reorganization and dose-sensitive activation of ground-state pluripotency.
Asunto(s)
Cromatina , Priones , Cromatina/genética , ADN/genética , Humanos , Proteína Homeótica Nanog/genética , Priones/genéticaRESUMEN
Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid-liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.
Asunto(s)
Reprogramación Celular , Cromatina/metabolismo , ADN/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , ADN/química , ADN/genética , Metilación de ADN , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/química , Factores de Transcripción de Tipo Kruppel/genética , Modelos Moleculares , Mutación , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción SOXB1/genética , Dedos de Zinc/genéticaRESUMEN
The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.
Asunto(s)
Adenosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas del Tejido Nervioso/genética , Factores de Empalme de ARN/genética , ARN/genética , Factores de Transcripción/genética , Adenosina/genética , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Humanos , Metilación , Elementos Reguladores de la Transcripción/genética , Activación Transcripcional/genéticaRESUMEN
Membrane-less organelles and RNP granules are enriched in RNA and RNA-binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear-cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C-terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid-liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane-less organelles, aggregation within phase-separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS-mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS-mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.
Asunto(s)
Ribonucleoproteína Nuclear Heterogénea A1/química , Proteínas Intrínsecamente Desordenadas/química , Agregado de Proteínas , Humanos , Dominios Proteicos , Electricidad EstáticaRESUMEN
Flaviviruses, including Zika, dengue, and West Nile viruses, are important human pathogens. The highly conserved NS2B-NS3 protease of Flavivirus is essential for viral replication and therefore a promising drug target. Through compound screening, followed by medicinal chemistry studies, a novel series of 2,5,6-trisubstituted pyrazine compounds are found to be potent, allosteric inhibitors of Zika virus protease (ZVpro) with IC50 values as low as 130 nM. Their structure-activity relationships are discussed. The ZVpro inhibitors also inhibit homologous proteases of dengue and West Nile viruses, and their inhibitory activities are correlated. The most potent compounds 47 and 103 potently inhibited Zika virus replication in cells with EC68 values of 300-600 nM and in a mouse model of Zika infection. These compounds represent novel pharmacological leads for drug development against Flavivirus infections.
Asunto(s)
Antivirales/uso terapéutico , Pirazinas/uso terapéutico , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/uso terapéutico , Proteínas Virales/metabolismo , Infección por el Virus Zika/tratamiento farmacológico , Regulación Alostérica/efectos de los fármacos , Animales , Antivirales/síntesis química , Línea Celular Tumoral , Virus del Dengue/enzimología , Humanos , Ratones , Estructura Molecular , Pirazinas/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Virus del Nilo Occidental/enzimología , Virus Zika/enzimologíaRESUMEN
Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HNRNPK. Losing these RNA-protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteostasis/genética , ARN Largo no Codificante/genética , Elementos de Nucleótido Esparcido Corto/genética , Apoptosis , Línea Celular , Citoplasma/metabolismo , Daño del ADN , Estrés del Retículo Endoplásmico , Activación Enzimática , Dosificación de Gen , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Mitosis , Modelos Biológicos , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de Secuencia/genética , eIF-2 QuinasaRESUMEN
Intrinsically disordered proteins as computationally predicted account for â¼1/3 of eukaryotic proteomes, are involved in a plethora of biological functions, and have been linked to several human diseases as a result of their dysfunctions. Here, we present a picture wherein an energetic continuum describes protein structural and conformational propensities, ranging from the hyperstable folded proteins on one end to the hyperdestabilized and sometimes functionally disordered proteins on the other. We distinguish between proteins that are folding-competent but disordered because of marginal stability and those that are disordered due mainly to the absence of folding code-completing structure-determining interactions, and postulate that disordered proteins that are unstructured by way of partial population of protein denatured states represent a sizable proportion of the proteome.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Ligandos , Conformación Proteica , Pliegue de Proteína , Proteoma/química , Proteoma/metabolismoRESUMEN
Influenza A virus (IAV) nonstructural protein 1 (NS1), a potent antagonist of the host immune response, is capable of interacting with RNA and a wide range of cellular proteins. NS1 consists of an RNA-binding domain (RBD) and an effector domain (ED) separated by a flexible linker region (LR). H5N1-NS1 has a characteristic 5-residue deletion in the LR, with either G (minor group) or E (major group) at the 71st position, and non-H5N1-NS1 contains E71 with an intact linker. Based on the orientation of the ED with respect to the RBD, previous crystallographic studies have shown that minor group H5N1-NS1(G71), a non-H5N1-NS1 [H6N6-NS1(E71)], and the LR deletion mutant H6N6-NS1(Δ80-84/E71) mimicking the major group H5N1-NS1 exhibit "open," "semiopen," and "closed" conformations, respectively, suggesting that NS1 exhibits a strain-dependent conformational preference. Here we report the first crystal structure of a naturally occurring H5N1-NS1(E71) and show that it adopts an open conformation similar to that of the minor group of H5N1-NS1 [H5N1-NS1(G71)]. We also show that H6N6-NS1(Δ80-84/E71) under a different crystallization condition and H6N6-NS1(Δ80-84/G71) also exhibit open conformations, suggesting that NS1 can adopt an open conformation irrespective of E or G at the 71st position. Our single-molecule fluorescence resonance energy transfer (FRET) analysis to investigate the conformational preference of NS1 in solution showed that all NS1 constructs predominantly exist in an open conformation. Further, our coimmunoprecipitation and binding studies showed that they all bind to cellular factors with similar affinities. Taken together, our studies suggest that NS1 exhibits strain-independent structural plasticity that allows it to interact with a wide variety of cellular ligands during viral infection.IMPORTANCE IAV is responsible for several pandemics over the last century and continues to infect millions annually. The frequent rise in drug-resistant strains necessitates exploring novel targets for developing antiviral drugs that can reduce the global burden of influenza infection. Because of its critical role in the replication and pathogenesis of IAV, nonstructural protein 1 (NS1) is a potential target for developing antivirals. Previous studies suggested that NS1 adopts strain-dependent "open," "semiopen," and "closed" conformations. Here we show, based on three crystal structures, that NS1 irrespective of strain differences can adopt an open conformation. We further show that NS1 from different strains primarily exists in an open conformation in solution and binds to cellular proteins with a similar affinity. Together, our findings suggest that conformational polymorphism facilitated by a flexible linker is intrinsic to NS1, and this may be the underlying factor allowing NS1 to bind several cellular factors during IAV replication.
Asunto(s)
Virus de la Influenza A/química , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Ligandos , Mutación , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Misfolding and aggregation of α-synuclein are linked to many neurodegenerative disorders, including Parkinson's and Alzheimer's disease. Despite intense research efforts, detailed structural characterization of early conformational transitions that initiate and drive α-synuclein aggregation remains elusive often due to the low sensitivity and ensemble averaging of commonly used techniques. Single-molecule Förster resonance energy transfer (smFRET) provides unique advantages in detecting minor conformations that initiate protein pathologic aggregation. In this chapter, we describe an smFRET-based method for characterizing early conformational conversions that are responsible for α-synuclein self-assembly and aggregation.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Agregación Patológica de Proteínas , Conformación Proteica , alfa-Sinucleína/química , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Pliegue de Proteína , Análisis Espectral , alfa-Sinucleína/metabolismoRESUMEN
Sox2 is a pioneer transcription factor that initiates cell fate reprogramming through locus-specific differential regulation. Mechanistically, it was assumed that Sox2 achieves its regulatory diversity via heterodimerization with partner transcription factors. Here, utilizing single-molecule fluorescence spectroscopy, we show that Sox2 alone can modulate DNA structural landscape in a dosage-dependent manner. We propose that such stoichiometric tuning of regulatory DNAs is crucial to the diverse biological functions of Sox2, and represents a generic mechanism of conferring functional plasticity and multiplicity to transcription factors.
Asunto(s)
ADN/química , Dominios HMG-Box , Conformación de Ácido Nucleico , Factores de Transcripción SOXB1/química , Imagen Individual de Molécula , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Regiones Promotoras Genéticas/genética , Unión ProteicaRESUMEN
Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of stress granules (SGs). These condensations are, however, usually assumed to be linked to pathologic fibrillation. Here, we show that physiologic condensation and pathologic fibrillation of RNPs are independent processes that can be unlinked with the chemical chaperone trimethylamine N-oxide (TMAO). Using the low-complexity disordered domain of the archetypical SG-protein TDP-43 as a model system, we show that TMAO enhances RNP liquid condensation yet inhibits protein fibrillation. Our results demonstrate effective decoupling of physiologic condensation from pathologic aggregation and suggest that selective targeting of protein fibrillation (without altering condensation) can be employed as a therapeutic strategy for RNP aggregation-associated degenerative disorders.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Extracción Líquido-Líquido , Metilaminas/química , Metilaminas/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Mutación , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteinopatías TDP-43/genética , Proteinopatías TDP-43/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
Neonatal rotavirus infections are predominantly asymptomatic. While an association with gastrointestinal symptoms has been described in some settings, factors influencing differences in clinical presentation are not well understood. Using multidisciplinary approaches, we show that a complex interplay between human milk oligosaccharides (HMOs), milk microbiome, and infant gut microbiome impacts neonatal rotavirus infections. Validating in vitro studies where HMOs are not decoy receptors for neonatal strain G10P[11], population studies show significantly higher levels of Lacto-N-tetraose (LNT), 2'-fucosyllactose (2'FL), and 6'-siallylactose (6'SL) in milk from mothers of rotavirus-positive neonates with gastrointestinal symptoms. Further, these HMOs correlate with abundance of Enterobacter/Klebsiella in maternal milk and infant stool. Specific HMOs also improve the infectivity of a neonatal strain-derived rotavirus vaccine. This study provides molecular and translational insight into host factors influencing neonatal rotavirus infections and identifies maternal components that could promote the performance of live, attenuated rotavirus vaccines.
Asunto(s)
Microbioma Gastrointestinal , Leche Humana/química , Leche Humana/microbiología , Oligosacáridos/metabolismo , Infecciones por Rotavirus/microbiología , Heces/microbiología , Humanos , Recién Nacido , Rotavirus/patogenicidad , Infecciones por Rotavirus/inmunología , Vacunas contra Rotavirus/inmunologíaRESUMEN
Rotaviruses (RVs) cause life-threatening diarrhea in infants and children worldwide. Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens (HBGA) as both cell attachment and susceptibility factors for the globally dominant P[4], P[6], and P[8] genotypes of human RVs. How these genotypes interact with HBGA is not known. Here, our crystal structures of P[4] and a neonate-specific P[6] VP8*s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs, consistent with their prevalence. Remarkably, the VP8* of P[6] RVs isolated from neonates displays subtle structural changes in this binding site that may restrict its ability to bind branched glycans. This provides a structural basis for the age-restricted tropism of some P[6] RVs as developmentally regulated unbranched glycans are more abundant in the neonatal gut.
Asunto(s)
Polisacáridos/metabolismo , Rotavirus/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Antígenos de Grupos Sanguíneos/biosíntesis , Línea Celular , Secuencia Conservada , Cristalografía por Rayos X , Fucosa/metabolismo , Humanos , Recién Nacido , Modelos Moleculares , Mutación/genética , Polisacáridos/química , Proteínas de Unión al ARN/química , Rotavirus/patogenicidad , Infecciones por Rotavirus/patología , Infecciones por Rotavirus/virología , Proteínas no Estructurales Virales/químicaRESUMEN
Neuropathological aggregates of the intrinsically disordered microtubule-associated protein Tau are hallmarks of Alzheimer’s disease, with decades of research devoted to studying the protein’s aggregation properties both in vitro and in vivo. Recent demonstrations that Tau is capable of undergoing liquid-liquid phase separation (LLPS) reveal the possibility that protein-enriched phase separated compartments could serve as initiation sites for Tau aggregation, as shown for other amyloidogenic proteins, such as the Fused in Sarcoma protein (FUS) and TAR DNA-binding protein-43 (TDP-43). Although truncation, mutation, and hyperphosphorylation have been shown to enhance Tau LLPS and aggregation, the effect of hyperacetylation on Tau aggregation remains unclear. Here, we investigate how the acetylation of Tau affects its potential to undergo phase separation and aggregation. Our data show that the hyperacetylation of Tau by p300 histone acetyltransferase (HAT) disfavors LLPS, inhibits heparin-induced aggregation, and impedes access to LLPS-initiated microtubule assembly. We propose that Tau acetylation prevents the toxic effects of LLPS-dependent aggregation but, nevertheless, contributes to Tau loss-of-function pathology by inhibiting Tau LLPS-mediated microtubule assembly.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Agregación Patológica de Proteínas/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Proteínas tau/metabolismo , Acetilación , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Heparina/química , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Extracción Líquido-Líquido , Microtúbulos/genética , Microtúbulos/metabolismo , Fosforilación , Agregación Patológica de Proteínas/genética , Factores de Transcripción p300-CBP/genética , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
Protein thermodynamic stability is intricately linked to cellular function, and altered stability can lead to dysfunction and disease. The linear extrapolation model (LEM) is commonly used to obtain protein unfolding free energies ([Formula: see text]) by extrapolation of solvent denaturation data to zero denaturant concentration. However, for some proteins, different denaturants result in non-coincident LEM-derived [Formula: see text] values, raising questions about the inherent assumption that the obtained [Formula: see text] values are intrinsic to the protein. Here, we used single-molecule FRET measurements to better understand such discrepancies by directly probing changes in the dimensions of the protein G B1 domain (GB1), a well-studied protein folding model, upon urea and guanidine hydrochloride denaturation. A comparison of the results for the two denaturants suggests denaturant-specific structural energetics in the GB1 denatured ensemble, revealing a role of the denatured state in the variable thermodynamic behavior of proteins.