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1.
Proc Natl Acad Sci U S A ; 117(39): 24234-24242, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32934141

RESUMO

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of atmospheric CO2 fixation by the biosphere. It catalyzes the addition of CO2 onto enolized ribulose 1,5-bisphosphate (RuBP), producing 3-phosphoglycerate which is then converted to sugars. The major problem of this reaction is competitive O2 addition, which forms a phosphorylated product (2-phosphoglycolate) that must be recycled by a series of biochemical reactions (photorespiratory metabolism). However, the way the enzyme activates O2 is still unknown. Here, we used isotope effects (with 2H, 25Mg, and 18O) to monitor O2 activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O2/CO2 selectivity. Neither the Rubisco form nor the use of solvent D2O and deuterated RuBP changed the 16O/18O isotope effect of O2 addition, in clear contrast with the 12C/13C isotope effect of CO2 addition. Furthermore, substitution of light magnesium (24Mg) by heavy, nuclear magnetic 25Mg had no effect on O2 addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O2) does not seem to be involved in O2 activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O2•-) production, must be insensitive to deuteration, and yields a predicted 16O/18O isotope effect and energy barrier close to observed values. Overall, O2 undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate.


Assuntos
Oxigênio/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Dióxido de Carbono/metabolismo , Transporte de Elétrons , Cinética , Isótopos de Oxigênio , Ozônio/metabolismo , Prótons
2.
Chem Rev ; 119(12): 6788-6821, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31017391

RESUMO

Allosteric signaling in biological molecules, which may be viewed as specific action at a distance due to localized perturbation upon binding of ligands or changes in environmental cues, is pervasive in biology. Insightful phenomenological Monod, Wyman, and Changeux (MWC) and Koshland, Nemethy, and Filmer (KNF) models galvanized research in describing allosteric transitions for over five decades, and these models continue to be the basis for describing the mechanisms of allostery in a bewildering array of systems. However, understanding allosteric signaling and the associated dynamics between distinct allosteric states at the molecular level is challenging and requires novel experiments complemented by computational studies. In this review, we first describe symmetry and rigidity as essential requirements for allosteric proteins or multisubunit structures. The general features, with MWC and KNF as two extreme scenarios, emerge when allosteric signaling is viewed from an energy landscape perspective. To go beyond the general theories, we describe computational tools that are either based solely on multiple sequences or their structures to predict the allostery wiring diagram. These methods could be used to predict the network of residues that carry allosteric signals. Methods to obtain molecular insights into the dynamics of allosteric transitions are briefly mentioned. The utility of the methods is illustrated by applications to systems ranging from monomeric proteins in which there is little conformational change in the transition between two allosteric states to membrane bound G-protein coupled receptors and multisubunit proteins. Finally, the role allostery plays in the functions of ATP-consuming molecular machines, bacterial chaperonin GroEL and molecular motors, is described. Although universal molecular principles governing allosteric signaling do not exist, we can draw the following general conclusions from a survey of different systems. (1) Multiple pathways connecting allosteric states are highly heterogeneous. (2) Allosteric signaling is exquisitely sensitive to the specific architecture of the system, which implies that the capacity for allostery is encoded in the structure itself. (3) The mechanical modes that connect distinct allosteric states are robust to sequence variations. (4) Extensive investigations of allostery in Hemoglobin and, more recently GroEL, show that to a large extent a network of salt bridge rearrangements serves as allosteric switches. In both these examples the dynamical changes in the allosteric switches are related to function.


Assuntos
Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Modelos Químicos , Proteínas/química , Proteínas/metabolismo , Regulação Alostérica , Conformação Proteica , Transdução de Sinais , Termodinâmica
3.
Proc Natl Acad Sci U S A ; 114(51): E10919-E10927, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29217641

RESUMO

Molecular chaperones facilitate the folding of proteins and RNA in vivo. Under physiological conditions, the in vitro folding of Tetrahymena ribozyme by the RNA chaperone CYT-19 behaves paradoxically; increasing the chaperone concentration reduces the yield of native ribozymes. In contrast, the protein chaperone GroEL works as expected; the yield of the native substrate increases with chaperone concentration. The discrepant chaperone-assisted ribozyme folding thus contradicts the expectation that it operates as an efficient annealing machine. To resolve this paradox, we propose a minimal stochastic model based on the Iterative Annealing Mechanism (IAM) that offers a unified description of chaperone-mediated folding of both proteins and RNA. Our theory provides a general relation that quantitatively predicts how the yield of native states depends on chaperone concentration. Although the absolute yield of native states decreases in the Tetrahymena ribozyme, the product of the folding rate and the steady-state native yield increases in both cases. By using energy from ATP hydrolysis, both CYT-19 and GroEL drive their substrate concentrations far out of equilibrium, thus maximizing the native yield in a short time. This also holds when the substrate concentration exceeds that of GroEL. Our findings satisfy the expectation that proteins and RNA be folded by chaperones on biologically relevant time scales, even if the final yield is lower than what equilibrium thermodynamics would dictate. The theory predicts that the quantity of chaperones in vivo has evolved to optimize native state production of the folded states of RNA and proteins in a given time.


Assuntos
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Algoritmos , Chaperonina 60/química , Chaperonina 60/metabolismo , Cinética , Modelos Moleculares , Conformação Molecular , Dobramento de Proteína , RNA/química , RNA/metabolismo , Dobramento de RNA , Relação Estrutura-Atividade , Especificidade por Substrato
4.
Proc Natl Acad Sci U S A ; 114(31): 8259-8264, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28710336

RESUMO

Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.


Assuntos
Chaperonina 60/química , Microscopia Crioeletrônica/métodos , Modelos Moleculares , Chaperonina 60/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
5.
Plant Cell Environ ; 41(4): 705-716, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29359811

RESUMO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most widespread carboxylating enzyme in autotrophic organisms. Its kinetic and structural properties have been intensively studied for more than half a century. Yet important aspects of the catalytic mechanism remain poorly understood, especially the oxygenase reaction. Because of its relatively modest turnover rate (a few catalytic events per second) and the competitive inhibition by oxygen, Rubisco is often viewed as an inefficient catalyst for CO2 fixation. Considerable efforts have been devoted to improving its catalytic efficiency, so far without success. In this review, we re-examine Rubisco's catalytic performance by comparison with other chemically related enzymes. We find that Rubisco is not especially slow. Furthermore, considering both the nature and the complexity of the chemical reaction, its kinetic properties are unremarkable. Although not unique to Rubisco, oxygenation is not systematically observed in enolate and enamine forming enzymes and cannot be considered as an inevitable consequence of the mechanism. It is more likely the result of a compromise between chemical and metabolic imperatives. We argue that a better description of Rubisco mechanism is still required to better understand the link between CO2 and O2 reactivity and the rationale of Rubisco diversification and evolution.


Assuntos
Ribulose-Bifosfato Carboxilase/metabolismo , Plantas/enzimologia , Plantas/metabolismo
6.
Proc Natl Acad Sci U S A ; 111(35): 12775-80, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25136110

RESUMO

The GroE chaperonins assist substrate protein (SP) folding by cycling through several conformational states. With each cycle the SP is, in turn, captured, unfolded, briefly encapsulated (t1/2 ∼ 1 s), and released by the chaperonin complex. The protein-folding functional form is the US-football-shaped GroEL:GroES2 complex. We report structures of two such "football" complexes to ∼ 3.7-Šresolution; one is empty whereas the other contains encapsulated SP in both chambers. Although encapsulated SP is not visible on the electron density map, using calibrated FRET and order-of-addition experiments we show that owing to SP-catalyzed ADP/ATP exchange both chambers of the football complex encapsulate SP efficiently only if the binding of SP precedes that of ATP. The two rings of GroEL thus behave as a parallel processing machine, rather than functioning alternately. Compared with the bullet-shaped GroEL:GroES1 complex, the GroEL:GroES2 football complex differs conformationally at the GroEL-GroES interface and also at the interface between the two GroEL rings. We propose that the electrostatic interactions between the ε-NH(3+) of K105 of helix D in one ring with the negatively charged carboxyl oxygen of A109 at the carboxyl end of helix D of the other ring provide the structural basis for negative inter-ring cooperativity.


Assuntos
Chaperonina 10/química , Chaperonina 60/química , Chaperoninas/química , Dobramento de Proteína , Thermus thermophilus/química , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Ligação de Hidrogênio , Modelos Químicos , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
7.
J Am Chem Soc ; 138(36): 11775-82, 2016 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-27532670

RESUMO

Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences ("helping hands") onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.


Assuntos
Proteínas/química , Proteínas/síntese química , Sequência de Aminoácidos , Técnicas de Química Sintética , Fluorenos/química , Dobramento de Proteína , Proteínas Ribossômicas/síntese química , Proteínas Ribossômicas/química , Solubilidade , Proteínas Virais/química
8.
Proc Natl Acad Sci U S A ; 110(27): E2451-9, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23723348

RESUMO

Noncovalently "stacked" tetramethylrhodamine (TMR) dimers have been used to both report and perturb the allosteric equilibrium in GroEL. A GroEL mutant (K242C) has been labeled with TMR, close to the peptide-binding site in the apical domain, such that TMR molecules on adjacent subunits are able to form dimers in the T allosteric state. Addition of ATP induces the transition to the R state and the separation of the peptide-binding sites, with concomitant unstacking of the TMR dimers. A statistical analysis of the spectra allowed us to compute the number and orientation of TMR dimers per ring as a function of the average number of TMR molecules per ring. The TMR dimers thus serve as quantitative reporter of the allosteric state of the system. The TMR dimers also serve as a surrogate for substrate protein, substituting in a more homogeneous, quantifiable manner for the heterogeneous intersubunit, intraring, noncovalent cross-links provided by the substrate protein. The characteristic stimulation of the ATPase activity by substrate protein is also mimicked by the TMR dimers. Using an expanded version of the nested cooperativity model, we determine values for the free energy of the TT to TR and TR to RR allosteric equilibria to be 27 ± 11 and 46 ± 2 kJ/mol, respectively. The free energy of unstacking of the TMR dimers was estimated at 2.6 ± 1.0 kJ/mol dimer. These results demonstrate that GroEL can perform work during the T to R transition, supporting the iterative annealing model of chaperonin function.


Assuntos
Chaperonina 60/química , Sítio Alostérico , Substituição de Aminoácidos , Fenômenos Biofísicos , Chaperonina 60/genética , Chaperonina 60/metabolismo , Corantes Fluorescentes , Modelos Moleculares , Sondas Moleculares , Mutagênese Sítio-Dirigida , Multimerização Proteica , Estrutura Quaternária de Proteína , Rodaminas , Processos Estocásticos
9.
Proc Natl Acad Sci U S A ; 110(46): E4289-97, 2013 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-24167257

RESUMO

The complex kinetics of Pi and ADP release by the chaperonin GroEL/GroES is influenced by the presence of unfolded substrate protein (SP). Without SP, the kinetics of Pi release are described by four phases: a "lag," a "burst" of ATP hydrolysis by the nascent cis ring, a "delay" caused by ADP release from the nascent trans ring, and steady-state ATP hydrolysis. The release of Pi precedes the release of ADP. The rate-determining step of the asymmetric cycle is the release of ADP from the trans ring of the GroEL-GroES1 "bullet" complex that is, consequently, the predominant species. In the asymmetric cycle, the two rings of GroEL function alternately, 180° out of phase. In the presence of SP, a change in the kinetic mechanism occurs. With SP present, the kinetics of ADP release are also described by four phases: a lag, a "surge" of ADP release attributable to SP-induced ADP/ATP exchange, and a "pause" during which symmetrical "football" particles are formed, followed by steady-state ATP hydrolysis. SP catalyzes ADP/ATP exchange on the trans ring. Now ADP release precedes the release of Pi, and the rate-determining step of the symmetric cycle becomes the hydrolysis of ATP by the symmetric GroEL-GroES2 football complex that is, consequently, the predominant species. A FRET-based analysis confirms that asymmetric GroEL-GroES1 bullets predominate in the absence of SP, whereas symmetric GroEL-GroES2 footballs predominate in the presence of SP. This evidence suggests that symmetrical football particles are the folding functional form of the chaperonin machine in vivo.


Assuntos
Trifosfato de Adenosina/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Complexos Multiproteicos/metabolismo , Dobramento de Proteína , Chaperonina 10/genética , Chaperonina 60/genética , Cumarínicos , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Cinética , Lactalbumina , Complexos Multiproteicos/genética , Mutação de Sentido Incorreto/genética , Proteínas de Ligação a Fosfato/metabolismo
10.
Proc Natl Acad Sci U S A ; 110(32): E2958-66, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23861496

RESUMO

The chaperonin proteins GroEL and GroES are cellular nanomachines driven by the hydrolysis of ATP that facilitate the folding of structurally diverse substrate proteins. In response to ligand binding, the subunits of a ring cycle in a concerted manner through a series of allosteric states (T, R, and R″), enabling work to be performed on the substrate protein. Removing two salt bridges that ordinarily break during the allosteric transitions of the WT permitted the structure of GroEL-ADP in the R state to be solved to 2.7 Šresolution. Whereas the equatorial domain displays almost perfect sevenfold symmetry, the apical domains, to which substrate proteins bind, and to a lesser extent, the intermediate domains display a remarkable asymmetry. Freed of intersubunit contacts, the apical domain of each subunit adopts a different conformation, suggesting a flexibility that permits interaction with diverse substrate proteins. This result contrasts with a previous cryo-EM study of a related allosteric ATP-bound state at lower resolution. After artificially imposing sevenfold symmetry it was concluded that a GroEL ring in the R-ATP state existed in six homogeneous but slightly different states. By imposing sevenfold symmetry on each of the subunits of the crystal structure of GroEL-ADP, we showed that the synthetic rings of (X-ray) GroEL-ADP and (cryo-EM) GroEL-ATP are structurally closely related. A deterministic model, the click stop mechanism, that implied temporal transitions between these states was proposed. Here, however, these conformational states are shown to exist as a structurally heterogeneous ensemble within a single ring.


Assuntos
Difosfato de Adenosina/química , Chaperonina 60/química , Conformação Proteica , Estrutura Terciária de Proteína , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Hidrólise , Modelos Moleculares , Mutação , Ligação Proteica
11.
Proc Natl Acad Sci U S A ; 110(46): E4298-305, 2013 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-24167279

RESUMO

Using calibrated FRET, we show that the simultaneous occupancy of both rings of GroEL by ATP and GroES occurs, leading to the rapid formation of symmetric GroEL:GroES2 "football" particles regardless of the presence or absence of substrate protein (SP). In the absence of SP, these symmetric particles revert to asymmetric GroEL:GroES1 "bullet" particles. The breakage of GroES symmetry requires the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry. These asymmetric particles are both persistent and dynamic; they turnover via the asymmetric cycle. When challenged with SP, however, they revert to symmetric particles within a second. In the presence of SP, the symmetric particles are also persistent and dynamic. They turn over via the symmetric cycle. Under these conditions, the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry also occur within the ensemble of particles. However, on account of SP-catalyzed ADP/ATP exchange, GroES symmetry is rapidly restored. The residence time of both GroES and SP on functional GroEL is reduced to ∼1 s, enabling many more iterations than was previously believed possible, consistent with the iterative annealing mechanism. This result is inconsistent with currently accepted models. Using a foldable SP, we show that as the SP folds to the native state and the population of unfolded SP declines, the population of symmetric particles reverts to asymmetric particles in parallel, a result that is consistent with the former being the folding functional form.


Assuntos
Trifosfato de Adenosina/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Dobramento de Proteína , Transferência Ressonante de Energia de Fluorescência , Guanosina/análogos & derivados , Hidrólise , Cinética , Tionucleosídeos
12.
Peptides ; 177: 171223, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38626843

RESUMO

Oxytocin (OXT), a neuropeptide consisting of only nine amino acids, is synthesized in the paraventricular and supraoptic nuclei of the hypothalamus. Although OXT is best known for its role in lactation and parturition, recent research has shown that it also has a significant impact on social behaviors in mammals. However, a comprehensive review of this topic is still lacking. In this paper, we systematically reviewed the effects of OXT on social behavior in mammals. These effects of OXT from the perspective of five key behavioral dimensions were summarized: parental behavior, anxiety, aggression, attachment, and empathy. To date, researchers have agreed that OXT plays a positive regulatory role in a wide range of social behaviors, but there have been controversially reported results. In this review, we have provided a detailed panorama of the role of OXT in social behavior and, for the first time, delved into the underlying regulatory mechanisms, which may help better understand the multifaceted role of OXT. Levels of OXT in previous human studies were also summarized to provide insights for diagnosis of mental disorders.


Assuntos
Ocitocina , Comportamento Social , Ocitocina/metabolismo , Ocitocina/fisiologia , Animais , Humanos , Neuropeptídeos/metabolismo , Mamíferos/metabolismo , Ansiedade/metabolismo , Ansiedade/psicologia , Agressão/fisiologia , Empatia/fisiologia , Feminino , Comportamento Materno/fisiologia
13.
Biology (Basel) ; 13(6)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38927253

RESUMO

Compared to pathogens Pseudomonas aeruginosa and P. putida, P. donghuensis HYS has stronger virulence towards Caenorhabditis elegans. However, the underlying mechanisms haven't been fully understood. The heme synthesis system is essential for Pseudomonas virulence, and former studies of HemN have focused on the synthesis of heme, while the relationship between HemN and Pseudomonas virulence were barely pursued. In this study, we hypothesized that hemN2 deficiency affected 7-hydroxytropolone (7-HT) biosynthesis and redox levels, thereby reducing bacterial virulence. There are four hemN genes in P. donghuensis HYS, and we reported for the first time that deletion of hemN2 significantly reduced the virulence of HYS towards C. elegans, whereas the reduction in virulence by the other three genes was not significant. Interestingly, hemN2 deletion significantly reduced colonization of P. donghuensis HYS in the gut of C. elegans. Further studies showed that HemN2 was regulated by GacS and participated in the virulence of P. donghuensis HYS towards C. elegans by mediating the synthesis of the virulence factor 7-HT. In addition, HemN2 and GacS regulated the virulence of P. donghuensis HYS by affecting antioxidant capacity and nitrative stress. In short, the findings that HemN2 was regulated by the Gac system and that it was involved in bacterial virulence via regulating 7-HT synthesis and redox levels were reported for the first time. These insights may enlighten further understanding of HemN-based virulence in the genus Pseudomonas.

14.
Biomed Pharmacother ; 172: 116191, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38320332

RESUMO

Folate receptor autoantibody (FRAA) has caught increasing attention since its discovery in biological fluids of patients with autism spectrum disorder (ASD), but quantification and understanding of its function are still in their infancy. In this study, we aimed to quantify serum binding-FRAA and explore its relation with serum folate, vitamin B12 (VB12) and ferritin. We quantitated serum binding-FRAA in 132 ASD children and 132 typically-developing (TD) children, as well as serum levels of folate, VB12 and ferritin. The results showed that serum binding-FRAA in the ASD group was significantly lower than that in the TD group (p < 0.0001). Further analysis showed that the difference between these two groups was attributed to boys in each group, not girls. There was no statistically significant difference in folate levels between the ASD and TD groups (p > 0.05). However, there was significant difference in boys between these two groups, not girls. Additionally, the combination of nitrite and binding-FRAA showed potential diagnostic value in patients with ASD (AUC > 0.7). Moreover, in the ASD group, the level of folate was consistent with that of binding-FRAA, whereas in the TD group, the binding-FRAA level was high when the folate level was low. Altogether, these differences revealed that the low serum FRAA in autistic children was mediated by multiple factors, which deserves more comprehensive investigation with larger population and mechanistic studies.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Masculino , Criança , Humanos , Ácido Fólico , Transtorno do Espectro Autista/epidemiologia , Autoanticorpos , Ferritinas
15.
Food Chem ; 405(Pt A): 134817, 2023 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-36370577

RESUMO

In order to extract sulforaphane (SFN) from broccoli via green and efficient ways, a novel method based on salting-out assisted deep eutectic solvent (DES) has been developed. Compared to known organic solvent- (such as dichloromethane, ethyl acetate, n-hexane, etc.) based liquid-liquid extraction, this new N8881Cl-based DES method exhibited excellent extraction efficiency for SFN, including a significant improvement due to the salting-out effect of KH2PO4. Under optimal conditions, 97.77 % of SFN was extracted by N8881Cl-EG DES and more than 82.5 % of SFN was recovered by activated carbon from DES. In addition, further studies with Kamlet-Taft parameters and density functional theory showed that the H-bond accepting capacity of hydrophobic DES, the existing vdW interaction, and the electrostatic interaction between N8881Cl-EG DES all contributed to efficient extraction of SFN. This is the first time that the underlying mechanism for SFN extraction by DES was revealed.


Assuntos
Brassica , Brassica/química , Solventes Eutéticos Profundos , Sulfóxidos , Isotiocianatos , Solventes/química , Cloreto de Sódio
16.
ACS Biomater Sci Eng ; 9(3): 1307-1319, 2023 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-36744996

RESUMO

Biomass carbon dots (CDs) derived from natural plants possess the advantages of low cost, photostability, and excellent biocompatibility, with potential applications in chemical sensing, bioimaging, and nanomedicine. However, the development of biomass CDs with excellent antioxidant activity and good biocompatibility is still a challenge. Herein, we propose a hypothesis for enhancing the antioxidant capacity of biomass CDs based on precursor optimization, extraction solvent, and other conditions with broccoli as the biomass. Compared to broccoli water extracts, broccoli powders, and broccoli organic solvent extracts, CDs derived from broccoli water extracts (BWE-CDs) have outstanding antioxidant properties due to the abundant C═C, carbonyl, and amino groups on their surface. After optimization of the preparation condition, the obtained BWE-CDs exhibit excellent free-radical scavenging activity with an EC50 of 68.2 µg/mL for DPPH• and 22.4 µg/mL for ABTS•+. Cytotoxicity and zebrafish embryotoxicity results indicated that BWE-CDs have lower cytotoxicity and better biocompatibility than that of CDs derived from organic solvents. In addition, BWE-CDs effectively scavenged reactive oxygen species (ROS) in A549 cells, 293T cells, and zebrafish, as well as eliminating inflammation in LPS-stimulated zebrafish. Mechanistic studies showed that the anti-inflammatory effect of BWE-CDs was dependent on the direct reaction of CDs with free radicals, the regulation of NO levels, and the upregulation of the expression of SOD and GPX-4. This work indicates that the antioxidant activity of CDs could be enhanced by using solvent extracts of biomass as precursors, and the obtained BWE-CDs exhibit characteristics of greenness, low toxicity, and excellent antioxidant and anti-inflammatory activities, which suggests the potential promising application of BWE-CDs as an antioxidant nanomedicine for inflammatory therapy.


Assuntos
Antioxidantes , Brassica , Animais , Peixe-Zebra , Carbono/química , Água , Anti-Inflamatórios/química , Solventes
17.
Front Mol Biosci ; 9: 1071168, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36479385

RESUMO

Chaperonins are biological nanomachines that help newly translated proteins to fold by rescuing them from kinetically trapped misfolded states. Protein folding assistance by the chaperonin machinery is obligatory in vivo for a subset of proteins in the bacterial proteome. Chaperonins are large oligomeric complexes, with unusual seven fold symmetry (group I) or eight/nine fold symmetry (group II), that form double-ring constructs, enclosing a central cavity that serves as the folding chamber. Dramatic large-scale conformational changes, that take place during ATP-driven cycles, allow chaperonins to bind misfolded proteins, encapsulate them into the expanded cavity and release them back into the cellular environment, regardless of whether they are folded or not. The theory associated with the iterative annealing mechanism, which incorporated the conformational free energy landscape description of protein folding, quantitatively explains most, if not all, the available data. Misfolded conformations are associated with low energy minima in a rugged energy landscape. Random disruptions of these low energy conformations result in higher free energy, less folded, conformations that can stochastically partition into the native state. Two distinct mechanisms of annealing action have been described. Group I chaperonins (GroEL homologues in eubacteria and endosymbiotic organelles), recognize a large number of misfolded proteins non-specifically and operate through highly coordinated cooperative motions. By contrast, the less well understood group II chaperonins (CCT in Eukarya and thermosome/TF55 in Archaea), assist a selected set of substrate proteins. Sequential conformational changes within a CCT ring are observed, perhaps promoting domain-by-domain substrate folding. Chaperonins are implicated in bacterial infection, autoimmune disease, as well as protein aggregation and degradation diseases. Understanding the chaperonin mechanism and the specific proteins they rescue during the cell cycle is important not only for the fundamental aspect of protein folding in the cellular environment, but also for effective therapeutic strategies.

18.
J Inorg Biochem ; 237: 111982, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36116154

RESUMO

Besides the canonical pathway of L-arginine oxidation to produce nitric oxide (NO) in vivo, the nitrate-nitrite-NO pathway has been widely accepted as another source for circulating NO in mammals, especially under hypoxia. To date, there have been at least ten heme-containing nitrite reductase-like proteins discovered in mammals with activities mainly identified in vitro, including four globins (hemoglobin, myoglobin, neuroglobin (Ngb), cytoglobin (Cygb)), three mitochondrial respiratory chain enzymes (cytochrome c oxidase, cytochrome bc1, cytochrome c), and three other heme proteins (endothelial nitric oxide synthase, cytochrome P450 and indoleamine 2,3-dioxygenase 1 (IDO1)). The pathophysiological functions of these proteins are closely related to their redox and spectroscopic properties, as well as their protein structure, although the physiological roles of Ngb, Cygb and IDO1 remain unclear. So far, comprehensive summaries of the redox and spectroscopic properties of these nitrite reductase-like hemoproteins are still lacking. In this review, we have mainly summarized the published data on the application of ultraviolet-visible, electron paramagnetic resonance, circular dichroism and resonance Raman spectroscopies, and X-ray crystallography in studying nitrite reductase-like activity of these 10 proteins, in order to sort out the relationships among enzymatic function, structure and spectroscopic characterization, which might help in understanding their roles in redox biology and medicine.


Assuntos
Proteínas do Tecido Nervoso , Nitrito Redutases , Animais , Nitrito Redutases/química , Proteínas do Tecido Nervoso/química , Globinas/química , Citoglobina/metabolismo , Oxirredução , Neuroglobina/metabolismo , Óxido Nítrico/química , Mamíferos/metabolismo
19.
Proc Natl Acad Sci U S A ; 105(45): 17339-44, 2008 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-18988739

RESUMO

In a study of the timing mechanism of the chaperonin nanomachine we show that the hemicycle time (HCT) is determined by the mean residence time (MRT) of GroES on the cis ring of GroEL. In turn, this is governed by allosteric interactions within the trans ring of GroEL. Ligands that enhance the R (relaxed) state (residual ADP, the product of the previous hemicycle, and K(+)) extend the MRT and the HCT, whereas ligands that enhance the T (taut) state (unfolded substrate protein, SP) decrease the MRT and the HCT. In the absence of SP, the chaperonin machine idles in the resting state, but in the presence of SP it operates close to the speed limit, set by the rate of ATP hydrolysis by the cis ring. Thus, the conformational states of the trans ring largely control the speed of the complete chaperonin cycle.


Assuntos
Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Potássio/metabolismo , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Cinética , Conformação Proteica , Fatores de Tempo
20.
Proc Natl Acad Sci U S A ; 105(45): 17334-8, 2008 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-18988745

RESUMO

The effects of potassium ion on the nested allostery of GroEL are due to increases in the affinity for nucleotide. Both positive allosteric transitions, TT-TR and TR-RR, occur at lower [ATP] as [K(+)] is increased. Negative cooperativity in the double-ringed system is also due to an increase in the affinity of the trans ring for the product ADP as [K(+)] is increased. Consequently, (i) rates of ATP hydrolysis are inversely proportional to [K(+)] and (ii) the residence time of GroES bound to the cis ring is prolonged and the hemicycle time extended. Substrate protein suppresses negative cooperativity by decreasing the affinity of the trans ring for ADP, reducing the hemicycle time to a constant minimum. The trans ring thus serves as a variable timer. ATP added to the asymmetric GroEL-GroES resting-state complex lacking trans ring ADP is hydrolyzed in the newly formed cis ring with a presteady-state burst of approximately 6 mol of Pi per mole of 14-mer. No burst is observed when the trans ring contains ADP. The amplitude and kinetics of ATP hydrolysis in the cis ring are independent of the presence or absence of encapsulated substrate protein and independent of K(+) at concentrations where there are profound effects on the linear steady-state rate. The hydrolysis of ATP by the cis ring constitutes a second, nonvariable timer of the chaperonin cycle.


Assuntos
Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Potássio/metabolismo , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Cinética , Conformação Proteica
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