RESUMO
The RNA-binding protein fused in sarcoma (FUS) forms ribonucleoprotein granules via liquid-liquid phase separation (LLPS) in the cytoplasm. The phase separation of FUS accelerates aberrant liquid-solid phase separation and leads to the onset of familial amyotrophic lateral sclerosis (ALS). We previously found that FUS forms two types of liquid condensates in equilibrium, specifically LP-LLPS (i.e., normal type) and HP-LLPS (i.e., aberrant type), each with different partial molar volumes. However, it is unclear how liquid condensates are converted to the pathogenic solid phase. Here, we report a mechanism underlying the aberrant liquid-to-solid phase transition of FUS liquid condensates and the inhibition of this transition with small molecules. We found that the liquid condensate formed via HP-LLPS had greatly reduced dynamics, which is a common feature of aged wild-type FUS droplets and the droplet-like assembly of the ALS patient-type FUS variant. The longer FUS remained on the HP-LLPS, the harder it was to transform it into a mixed state (i.e., one-phase). These results indicate that liquid-to-solid phase transition, namely the aging of droplets, is accelerated with HP-LLPS. Interestingly, arginine suppressed the aging of droplets and HP-LLPS formation more strongly than LP-LLPS formation. These data indicate that the formation of HP-LLPS via the one-phase state or LP-LLPS is a pathway leading to irreversible solid aggregates. Dopamine and pyrocatechol also suppressed HP-LLPS formation. Our data highlight the potential of HP-LLPS to be used as a therapeutic target and arginine as a plausible drug candidate for ALS-causing FUS.
Assuntos
Esclerose Lateral Amiotrófica , Sarcoma , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Arginina , Humanos , Transição de Fase , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismoRESUMO
Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 calâ Å-3.
Assuntos
Proteínas/química , Bacteriófago T4/química , Muramidase/química , Ressonância Magnética Nuclear Biomolecular/métodos , Pressão , Conformação Proteica , Desnaturação Proteica , Dobramento de ProteínaRESUMO
The RNA-binding protein fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) both in vivo and in vitro. Self-assembled liquid droplets of FUS transform into reversible hydrogels and into more irreversible and toxic aggregates. Although LLPS can be a precursor of irreversible aggregates, a generic method to study kinetics of the formation of LLPS has not been developed. Here, we demonstrated the pressure-jump kinetics of phase transition between the 1-phase state and FUS-LLPS states observed at low pressure (<2 kbar, LP-LLPS) and high pressure (>2 kbar, HP-LLPS) using high-pressure UV/vis spectroscopy. Absorbance (turbidity) changes were reproduced repeatedly using pressure cycles. The Johnson-Mehl-Avrami-Kolmogorov theory was used to understand droplet formation occurring via nucleation and growth. The Avrami exponent n, representing the dimensionality of growing droplets, and the reaction rate constant k were calculated. The HP-LLPS formation rate was â¼2-fold slower than that of LP-LLPS. The Avrami exponent obtained for both LLPS states could be explained by diffusion-limited growth. Nucleation and growth rates decreased during LP-LLPS formation (n = 0.51), and the nucleation rate decreased with a constant growth rate in HP-LLPS formation (n = 1.4). The HP-LLPS vanishing rate was â¼20-fold slower than that of LP-LLPS. This difference in vanishing rates indicates a stronger intermolecular interaction in HP-LLPS than in LP-LLPS, which might promote transformation into irreversible aggregates in the droplets. Further, direct transition from HP-LLPS to LP-LLPS was observed. This indicates that interconversion between LP-LLPS and HP-LLPS occurs in equilibrium. Formation of reversible liquid droplets, followed by phase transition into another liquid phase, could thus be part of the physiological maturation process of FUS-LLPS.
Assuntos
Proteína FUS de Ligação a RNA/metabolismo , Cinética , Transição de Fase , Pressão , Multimerização Proteica , Proteína FUS de Ligação a RNA/químicaRESUMO
Structural characterization of alternatively folded and partially disordered protein conformations remains challenging. Outer surface protein A (OspA) is a pivotal protein in Borrelia infection, which is the etiological agent of Lyme disease. OspA exists in equilibrium with intermediate conformations, in which the central and the C-terminal regions of the protein have lower stabilities than the N-terminal. Here, we characterize pressure- and temperature-stabilized intermediates of OspA by nuclear magnetic resonance spectroscopy combined with paramagnetic relaxation enhancement (PRE). We found that although the C-terminal region of the intermediate was partially disordered, it retains weak specific contact with the N-terminal region, owing to a twist of the central ß-sheet and increased flexibility in the polypeptide chain. The disordered C-terminal region of the pressure-stabilized intermediate was more compact than that of the temperature-stabilized form. Further, molecular dynamics simulation demonstrated that temperature-induced disordering of the ß-sheet was initiated at the C-terminal region and continued through to the central region. An ensemble of simulation snapshots qualitatively described the PRE data from the intermediate and indicated that the intermediate structures of OspA may expose tick receptor-binding sites more readily than does the basic folded conformation.
Assuntos
Antígenos de Superfície/química , Proteínas de Artrópodes/química , Proteínas da Membrana Bacteriana Externa/química , Vacinas Bacterianas/química , Borrelia/química , Proteínas Intrinsicamente Desordenadas/química , Lipoproteínas/química , Receptores de Superfície Celular/química , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas de Artrópodes/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas/genética , Vacinas Bacterianas/metabolismo , Sítios de Ligação , Borrelia/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Carrapatos/microbiologiaRESUMO
Adeno-associated virus (AAV) has been studied as a safe delivery tool for gene therapy of retinal blinding diseases such as Leber's congenital amaurosis (LCA). The tropism of recombinant AAV (rAAV) including its specificity and efficiency in targeting retinal cell types has been studied with native or engineered capsids, along with specific promoters. However, one of the rAAV serotypes, rAAV2/6, has not been well-studied based on a report of low infection efficiency in the retina. We investigated the tropism of several rAAVs by subretinal injection in the adult mouse and found that rAAV2/6 predominantly infected cone photoreceptors including the main spectral type. Our data suggest that subretinal injection with rAAV2/6 may provide both an efficacious and specific means of gene delivery to cone photoreceptors in murine retinas.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Doenças Retinianas/terapia , Animais , Vetores Genéticos/administração & dosagem , Injeções , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Camundongos da Linhagem 129 , Opsinas/genética , Opsinas/metabolismo , Retina/virologia , Células Fotorreceptoras Retinianas Cones/virologia , Doenças Retinianas/genética , Resultado do TratamentoRESUMO
Conformational fluctuations of proteins are crucially important for their functions. However, changes in the location and dynamics of hydrated water in many proteins accompanied by the conformational transition have not been fully understood. Here, we used phase-modulated clean chemical exchange NMR approach to investigate pressure-induced changes in water-to-amide proton exchange occurring at sub-second time scale. With the transition of ubiquitin from its native conformation (N1) to an alternative conformation (N2) at 250 MPa, proton exchange rates of residues 32-35, 40-41, and 71, which are located at the C-terminal side of the protein, were significantly increased. These observations can be explained by the destabilization of the hydrogen bonds in the backbone and partial exposure of those amide groups to solvent in N2. We conclude that phase-modulated clean chemical exchange NMR approach coupled with pressure perturbation will be a useful tool for investigations of more open and hydrated protein structures.
Assuntos
Ubiquitina/química , Ubiquitina/metabolismo , Água/metabolismo , Amidas/química , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O2-binding sites by O2-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer ß-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O2-induced PREs for amide protons located around the largest cavity and at the central ß-sheet. We suggested three potential O2-accessible sites in the protein based on the 1/r6 distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central ß-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O2 generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O2 within the largest cavity, egress of O2 from the cavity, and ingress of O2 in the surface crevice of the ß-sheet. These molecular dynamics simulation results qualitatively explained the O2-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas/metabolismo , Lipoproteínas/metabolismo , Oxigênio/metabolismo , Antígenos de Superfície/química , Proteínas da Membrana Bacteriana Externa/química , Vacinas Bacterianas/química , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Lipoproteínas/química , Simulação de Dinâmica Molecular , Movimento (Física) , Dinâmica não Linear , Ressonância Magnética Nuclear Biomolecular , Oxigênio/química , Estrutura Secundária de ProteínaRESUMO
Rational mutation of proteins based on their structural and dynamic characteristics is a useful strategy for amplifying specific fluctuations in proteins. Here, we show the effects of mutation on the conformational fluctuations and thermodynamic stability of ubiquitin. In particular, we focus on the salt bridge between K11 and E34 and the hydrogen bond between I36 and Q41, which are predicted to control the fluctuation between the basic folded state, N1, and the alternatively folded state, N2, of the protein, using high-pressure NMR spectroscopy. The E34A mutation, which disrupts the salt bridge, did not alter picosecond-to-nanosecond, microsecond-to-millisecond dynamic motions, and stability of the protein, while the Q41N mutation, which destabilizes the hydrogen bond, specifically amplified the N1-N2 conformational fluctuation and decreased stability. Based on the observed thermodynamic stabilities of the various conformational states, we showed that in the Q41N mutant, the N1 state is more significantly destabilized than the N2 state, resulting in an increase in the relative population of N2. Identifying the interactions controlling specific motions of a protein will facilitate molecular design to achieve functional dynamics beyond native state dynamics.
Assuntos
Ubiquitina/química , Ubiquitina/genética , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Conformação Proteica , Estabilidade Proteica , TermodinâmicaRESUMO
Hydrogen exchange (HX) NMR spectroscopy is widely used for monitoring structure, stability and dynamics of proteins at the level of individual residues. The stochastic replacement of protons by deuterons typically leads to an exponential decrease of the NMR signals. However, an unusual signal increase was observed in HX of several amides for T4 lysozyme L99A. This effect can be attributed to peak sharpening as a result of reduced dipolar relaxation from proximal amide protons that experience more rapid hydrogen/deuterium (H/D) exchange. The behavior was specifically observed at the termini of secondary structure elements, where large differences in protection against H/D exchange are observed. This effect is expected to be more widespread in NMR HX studies, and is important for the accurate determination of protection factors.
Assuntos
Medição da Troca de Deutério , Hidrogênio/metabolismo , Espectroscopia de Ressonância Magnética , Processamento de Sinais Assistido por Computador , Ligação de Hidrogênio , Modelos BiológicosRESUMO
Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change in the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence.
Assuntos
Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Pressão , Ubiquitina/química , Ácido Bromídrico , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Solventes/química , Água/químicaRESUMO
High-pressure nuclear magnetic resonance (NMR) spectroscopy has revealed that ubiquitin has at least two high-energy states--an alternatively folded state N2 and a locally disordered state I--between the basic folded state N1 and totally unfolded U state. The high-energy states are conserved among ubiquitin-like post-translational modifiers, ubiquitin, NEDD8, and SUMO-2, showing the E1-E2-E3 cascade reaction. It is quite intriguing that structurally similar high-energy states are evolutionally conserved in the ubiquitin-like modifiers, and the thermodynamic stabilities vary among the proteins. To investigate atomic details of the high-energy states, a Q41N mutant of ubiquitin was created as a structural model of N2, which is 71% populated even at atmospheric pressure. The convergent structure of the "pure" N2 state was obtained by nuclear Overhauser effect (NOE)-based structural analysis of the Q41N mutant at 2.5 kbar, where the N2 state is 97% populated. The N2 state of ubiquitin is closely similar to the conformation of the protein bound to the ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is best explained by conformational selection rather than by induced-fit motion.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Ubiquitina/química , Pressão Atmosférica , Dobramento de ProteínaRESUMO
Although the structure, function, conformational dynamics, and controlled thermodynamics of proteins are manifested by their corresponding amino acid sequences, the natural rules for molecular design and their corresponding interplay remain obscure. In this study, we focused on the role of internal cavities of proteins in conformational dynamics. We investigated the pressure-induced responses from the cavity-enlarged L99A mutant of T4 lysozyme, using high-pressure NMR spectroscopy. The signal intensities of the methyl groups in the (1)H/(13)C heteronuclear single quantum correlation spectra, particularly those around the enlarged cavity, decreased with the increasing pressure, and disappeared at 200 MPa, without the appearance of new resonances, thus indicating the presence of heterogeneous conformations around the cavity within the ground state ensemble. Above 200 MPa, the signal intensities of >20 methyl groups gradually decreased with the increasing pressure, without the appearance of new resonances. Interestingly, these residues closely matched those sensing a large conformational change between the ground- and high-energy states, at atmospheric pressure. (13)C and (1)H NMR line-shape simulations showed that the pressure-induced loss in the peak intensity could be explained by the increase in the high-energy state population. In this high-energy state, the aromatic side chain of F114 gets flipped into the enlarged cavity. The accommodation of the phenylalanine ring into the efficiently packed cavity may decrease the partial molar volume of the high-energy state, relative to the ground state. We suggest that the enlarged cavity is involved in the conformational transition to high-energy states and in the volume fluctuation of the ground state.
Assuntos
Bacteriófago T4 , Muramidase/química , Proteínas Virais/química , Isótopos de Carbono , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Muramidase/genética , Mutação , Ressonância Magnética Nuclear Biomolecular , Pressão , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Virais/genética , Água/químicaRESUMO
We present the nuclear Overhauser effect-based structure determination of the Q41N variant of ubiquitin at 2500 bar, where the alternatively folded N2 state is 97% populated. This allows us to characterize the structure of the "pure" N2 state of ubiquitin. The N2 state shows a substantial change in the orientation of strand ß5 compared to that of the normal folded N1 state, which matches the changes seen upon binding of ubiquitin to ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is therefore best explained by conformational selection rather than induced-fit motion.
Assuntos
Dobramento de Proteína , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant ß-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.
Assuntos
Amiloide , Esclerose Lateral Amiotrófica , Corpos de Inclusão Intranuclear , Neurônios , Dobramento de Proteína , Motivos de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HEK293 , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Corpos de Inclusão Intranuclear/patologia , Espectroscopia de Ressonância Magnética , Masculino , Neurônios/metabolismo , Neurônios/patologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Splicing de RNA , UbiquitinaçãoRESUMO
To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.
Assuntos
Substituição de Aminoácidos , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Mutação de Sentido Incorreto , Tetra-Hidrofolato Desidrogenase/química , Compostos de Bário/química , Domínio Catalítico , Cloretos/química , Estabilidade Enzimática/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Concentração de Íons de Hidrogênio , Solventes/química , Especificidade por Substrato , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
We examined the effects of trimethylamine N-oxide (TMAO) and urea (known osmolytes) on the liquid-liquid phase separation (LLPS) of fused in sarcoma (FUS) and three FUS-LLPS states: LLPS states at atmospheric pressure with low- and high-salt concentrations and a re-entrant LLPS state above 2 kbar. Temperature- and pressure-scan turbidity measurements revealed that TMAO and urea contributed to stabilizing and destabilizing LLPS, respectively. These results can be attributed to the excluded volume effect of TMAO (preferential hydration) and preferential interaction of urea with proteins. Additionally, TMAO counteracted the effects of equimolar urea on LLPS, a phenomenon not previously reported. The concept of the m-value for osmolyte-induced protein folding and unfolding can be applied to the osmolyte's effects on LLPS. In conclusion, biomolecular LLPS can be modulated by preferential hydration and the interaction of small osmolytes with proteins, thereby facilitating LLPS formation, even in extreme environments characterized by high-salt, high-urea, and high-pressure conditions.
Assuntos
Metilaminas , Ureia , Metilaminas/química , Ureia/química , Água/química , Dobramento de Proteína , Proteínas/química , Temperatura , Separação de FasesRESUMO
Intracellular aggregation of fused in sarcoma (FUS) is associated with the pathogenesis of familial amyotrophic lateral sclerosis (ALS). Under stress, FUS forms liquid droplets via liquid-liquid phase separation (LLPS). Two types of wild-type FUS LLPS exist in equilibrium: low-pressure LLPS (LP-LLPS) and high-pressure LLPS (HP-LLPS); the former dominates below 2 kbar and the latter over 2 kbar. Although several disease-type FUS variants have been identified, the molecular mechanism underlying accelerated cytoplasmic granule formation in ALS patients remains poorly understood. Herein, we report the reversible formation of the two LLPS states and the irreversible liquid-solid transition, namely droplet aging, of the ALS patient-type FUS variant R495X using fluorescence microscopy and ultraviolet-visible absorption spectroscopy combined with perturbations in pressure and temperature. Liquid-to-solid phase transition was accelerated in the HP-LLPS of R495X than in the wild-type variant; arginine slowed the aging of droplets at atmospheric conditions by inhibiting the formation of HP-LLPS more selectively compared to that of LP-LLPS. Our findings provide new insight into the mechanism by which R495X readily forms cytoplasmic aggregates. Targeting the aberrantly formed liquid droplets (the HP-LLPS state) of proteins with minimal impact on physiological functions could be a novel therapeutic strategy for LLPS-mediated protein diseases.
Assuntos
Esclerose Lateral Amiotrófica , Proteína FUS de Ligação a RNA , Sarcoma , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Transição de Fase , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismoRESUMO
To improve bladder compliance in patients with low-compliance bladders, augmentation cystoplasty with the intestinal tract is performed. However, the use of the intestinal tract often leads to serious surgical complications. Tissue engineering technologies have the potential to improve bladder compliance without using the intestinal tract. In this study, we fabricated bi-layered adipose-derived mesenchymal cell (AMC) sheets and then determined whether the bi-layered AMC sheets could improve bladder compliance in rats with spinal cord injury (SCI). The abdominal adipose tissues of green fluorescence protein (GFP)-transfected Sprague-Dawley (SD) rats were harvested, and the attached and proliferating cells on type I collagen were used as AMCs. The AMCs were then cultured on temperature-responsive culture dishes. After reaching over-confluence, the AMCs that maintained cell-cell contacts were detached from the dishes and applied to a gelatin hydrogel sheet. Then, another detached AMC monolayer was accumulated on the AMC monolayer-applied gelatin. Prior to 4 weeks of transplantation, the levels of T8-9 in the spinal cords of recipient SD rats were partially transected. After producing the bi-layered AMC sheets and the rats with SCI, the detrusor muscles of the anterior bladder walls of the rats with SCI were incised, and the bi-layered AMC sheet was patch-transplanted onto the exposed bladder epithelium (n = 8). As a control, the sham operation was performed (n = 7). Four weeks after the transplantation, bladder capacity and bladder compliance in AMC sheet-transplanted SCI rats were significantly higher than those in sham-operated control SCI rats. The smooth muscle layers in AMC sheet-transplanted bladders were significantly larger than those in control bladders. In addition, the collagen fibers in the AMC sheet-transplanted bladders were significantly smaller than those in the control bladders. Some GFP-positive transplanted AMCs differentiated into smooth muscle actin- or desmin-positive cells. Furthermore, GFP-positive cells secreted transforming growth factor-ß1 or vascular endothelial growth factor. Therefore, this study showed that bi-layered AMC sheets could improve bladder compliance and bladder tissues in SCI rats.
RESUMO
It is becoming increasingly clear that proteins transiently populate high-energy excited states as a necessary requirement for function. Here, we demonstrate that rational mutation based on the characteristics of the structure and dynamics of proteins obtained from pressure experiments is a new strategy for amplifying particular fluctuations in proteins. We have previously shown that ubiquitin populates a high-energy conformer, N2, at high pressures. Here, we show that the Q41N mutation favors N2: high-pressure nuclear magnetic resonance (NMR) shows that N2 is â¼70% populated in Q41N but only â¼20% populated in the wild type at ambient pressure. This allows us to characterize the structure of N2, in which α1-helix, the following loop, ß3-strand, and ß5-strand change their orientations relative to the remaining regions. Conformational fluctuation on the microsecond time scale, characterized by (15)N spin relaxation NMR analysis, is markedly increased for these regions of the mutant. The N2 conformers produced by high pressure and by the Q41N mutation are quite similar in both structure and dynamics. The conformational change to produce N2 is proposed to be a novel dynamic feature beyond the known recognition dynamics of the protein. Indeed, it is orthogonal to that seen when proteins containing a ubiquitin-interacting motif bind at the hydrophobic patch of ubiquitin but matches changes seen on binding to the E2 conjugating enzyme. More generally, structural and dynamic effects of hydrodynamic pressure are shown to be useful for characterizing functionally important intermediates.
Assuntos
Dobramento de Proteína , Ubiquitina/química , Ubiquitina/genética , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Conformação Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
The unique role of pressure in protein folding studies is emphasized. Variable-pressure NMR experiments carried out under equilibrium conditions give unique opportunities to explore the energy landscape for protein folding. Intermediate conformers that may appear transiently in the kinetic folding experiments may be stably trapped under pressure, allowing examination of their conformations in site-specific detail with modern NMR spectroscopy. The intimate relationship between the kinetic folding experiment and the equilibrium pressure experiment is described with examples from ubiquitin and hen lysozyme.