RESUMO
G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gßγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gßγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.
Assuntos
Guanosina Trifosfato , Proteínas Heterotriméricas de Ligação ao GTP , Simulação de Dinâmica Molecular , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Regulação Alostérica , Motivos de Aminoácidos , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.
Assuntos
Apoptose/fisiologia , Inflamação/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Humanos , Imunidade Inata/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Serina/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismoRESUMO
Alzheimer's disease is a neurodegenerative disorder that causes age-dependent neurological and cognitive declines. The treatments for Alzheimer's disease pose a significant challenge, because the mechanisms of disease are not being fully understood. Malfunction of the blood-brain barrier is increasingly recognized as a major contributor to the pathophysiology of Alzheimer's disease, especially at the early stages of the disease. However, the underlying mechanisms remain poorly characterized, while few molecules can directly target and improve blood-brain barrier function in the context of Alzheimer's disease. Here, we showed dysfunctional blood-brain barrier in patients with Alzheimer's disease reflected by perivascular accumulation of blood-derived fibrinogen in the hippocampus and cortex, accompanied by decreased tight junction proteins Claudin-5 and glucose transporter Glut-1 in the brain endothelial cells. In the APPswe/PS1dE9 (APP/PS1) mouse model of Alzheimer's disease, blood-brain barrier dysfunction started at 4 months of age and became severe at 9 months of age. In the cerebral microvessels of APP/PS1 mice and amyloid-ß-treated brain endothelial cells, we found suppressed Wnt/ß-catenin signalling triggered by an increase of GSK3ß activation, but not an inhibition of the AKT pathway or switching to the Wnt/planar cell polarity pathway. Furthermore, using our newly developed optogenetic tool for controlled regulation of LRP6 (upstream regulator of the Wnt signalling) to activate Wnt/ß-catenin pathway, blood-brain barrier malfunction was restored by preventing amyloid-ß-induced brain endothelial cells impairments and promoting the barrier repair. In conclusion, targeting LRP6 in the Wnt/ß-catenin pathway in the brain endothelium can alleviate blood-brain barrier malfunction induced by amyloid-ß, which may be a potential treatment strategy for Alzheimer's disease.
Assuntos
Doença de Alzheimer , Camundongos , Animais , Doença de Alzheimer/metabolismo , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , beta Catenina , Peptídeos beta-Amiloides/metabolismo , Via de Sinalização Wnt , Modelos Animais de Doenças , Camundongos TransgênicosRESUMO
All cells respond to osmotic stress by implementing molecular signaling events to protect the organism. Failure to properly adapt can lead to pathologies such as hypertension and ischemia-reperfusion injury. Mitogen-activated protein kinases (MAPKs) are activated in response to osmotic stress, as well as by signals acting through G protein-coupled receptors (GPCRs). For proper adaptation, the action of these kinases must be coordinated. To identify second messengers of stress adaptation, we conducted a mass spectrometry-based global metabolomics profiling analysis, quantifying nearly 300 metabolites in the yeast S. cerevisiae. We show that three branched-chain amino acid (BCAA) metabolites increase in response to osmotic stress and require the MAPK Hog1. Ectopic addition of these BCAA derivatives promotes phosphorylation of the G protein α subunit and dampens G protein-dependent transcription, similar to that seen in response to osmotic stress. Conversely, genetic ablation of Hog1 activity or the BCAA-regulatory enzymes leads to diminished phosphorylation of Gα and increased transcription. Taken together, our results define a new class of candidate second messengers that mediate cross talk between osmotic stress and GPCR signaling pathways.
Assuntos
Aminoácidos/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Pressão Osmótica , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Regulação Fúngica da Expressão Gênica , Metaboloma , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drug-receptor interaction analysis and ligand screening. In our example assay using beta2-adrenergic receptor (ß2-AR), the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of ß2-AR on the resin. The immobilized ß2-AR as a stationary phase enabled us to rapidly determine the binding of four drugs to ß2-AR. By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting ß2-AR in Fructus Perillae. We concluded that O6-benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.
Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Descoberta de Drogas , Guanina/análogos & derivados , Ensaios de Triagem em Larga Escala , Receptores Adrenérgicos beta 2/metabolismo , Cinamatos/química , Depsídeos/química , Guanina/química , Guanina/metabolismo , Humanos , Ligantes , Perilla/química , Receptores Adrenérgicos beta 2/análise , Propriedades de Superfície , Ácido RosmarínicoRESUMO
The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Células Cultivadas , Humanos , Hidrólise , Camundongos , Modelos Moleculares , Células NIH 3T3 , Mutação Puntual , Conformação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Transdução de SinaisRESUMO
Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms.
Assuntos
Mutação , Saccharomyces cerevisiae/genética , Serina/genética , alfa-Sinucleína/genética , Animais , Western Blotting , Encéfalo/metabolismo , Caseína Quinase I/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Hipocampo/metabolismo , Humanos , Cinética , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , Doença de Parkinson/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Especificidade por Substrato , alfa-Sinucleína/metabolismoRESUMO
Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments. An ability to specifically and selectively control protein function via conformational modulation is an important goal for development of novel therapeutics and studies of protein mechanism in biological networks and disease. Here we applied a second-harmonic generation-based technique for studying protein conformation in solution and in real time to the intrinsically disordered, Parkinson disease related protein α-synuclein. From a fragment library, we identified small molecule modulators that bind to monomeric α-synuclein in vitro and significantly reduce α-synuclein aggregation in a neuronal cell culture model. Our results indicate that the conformation of α-synuclein is linked to the aggregation of protein in cells. They also provide support for a therapeutic strategy of targeting specific conformations of the protein to suppress or control its aggregation.
Assuntos
Antiparkinsonianos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , alfa-Sinucleína/química , Antiparkinsonianos/química , Antiparkinsonianos/isolamento & purificação , Linhagem Celular Tumoral , Humanos , Ligantes , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/isolamento & purificaçãoRESUMO
Alpha-synuclein (αS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS-Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.
Assuntos
Proteínas de Drosophila/metabolismo , GTP Fosfo-Hidrolases/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Escherichia coli , GTP Fosfo-Hidrolases/genética , Humanos , Camundongos , Modelos Moleculares , Transtornos dos Movimentos/fisiopatologia , Mutação , Neurônios/fisiologia , Fosforilação , Ligação Proteica , Ratos , Sinaptossomos/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Transition metal (TM) single-atom catalysts (SACs) have been widely applied in photocatalytic CO2 reduction. In this work, n-p codoping engineering is introduced to account for the modulation of photocatalytic CO2 reduction on a two-dimensional (2D) bismuth-oxyhalide-based cathode by using first-principles calculation. n-p codoping is established via the Coulomb interactions between the negatively charged TM SACs and the positively charged Cl vacancy (VCl) in the dopant-defect pairs. Based on the formation energy of charged defects, neutral dopant-defect pairs for the Fe, Co, and Ni SACs (PTM0) and the -1e charge state of the Cu SAC-based pair (PCu-1) are stable. The electrostatic attraction of the n-p codoping strengthens the stability and solubility of TM SACs by neutralizing the oppositely charged VCl defect and TM dopant. The n-p codoping stabilizes the electron accumulation around the TM SACs. Accumulated electrons modify the d-orbital alignment and shift the d-band center toward the Fermi level, enhancing the reducing capacity of TM SACs based on the d-band theory. Besides the electrostatic attraction of the n-p codoping, the PCu-1 also accumulates additional electrons surrounding Cu SACs and forms a half-occupied dx2-y2 state, which further upshifts the d-band center and improves photocatalytic CO2 reduction. The metastability of Cl multivacancies limits the concentration of the n-p pairs with Cl multivacancies (PTM@nCl (n > 1)). Positively charged centers around the PTM@nCl (n > 1) hinders the CO2 reduction by shielding the charge transfer to the CO2 molecule.
RESUMO
Heterotrimeric G proteins (Gα, Gß and Gγ) act downstream of G-protein-coupled receptors (GPCRs) to mediate signaling pathways that regulate various physiological processes and human disease conditions. Previously, human Gαi and its yeast homolog Gpa1 have been reported to function as intracellular pH sensors, yet the pH sensing capabilities of Gαi and the underlying mechanism remain to be established. Herein, we identify a pH sensing network within Gαi, and evaluate the consequences of pH modulation on the structure and stability of the G-protein. We find that changes over the physiological pH range significantly alter the structure and stability of Gαi-GDP, with the protein undergoing a disorder-to-order transition as the pH is raised from 6.8 to 7.5. Further, we find that modulation of intracellular pH in HEK293 cells regulates Gαi-Gßγ release. Identification of key residues in the pH-sensing network allowed the generation of low pH mimetics that attenuate Gαi-Gßγ release. Our findings, taken together, indicate that pH-dependent structural changes in Gαi alter the agonist-mediated Gßγ dissociation necessary for proper signaling.
RESUMO
High pressure can change the valence electron arrangement of the elements, and it can be as a new method for the emergence of unexpected new compounds. In this paper, the Ca-Ar compounds at 0-200 GPa are systematically investigated by using CALYPSO structure prediction methods combined with first principles calculations. The study of the Ca-Ar system can provide theoretical guidance for the exploration of new structures of inert elemental Ar compounds under high pressure. A stable structure:P63/mmc-CaAr and six metastable structures:Rm-CaAr2,P4/mmm-CaAr2,Pm1-CaAr3,P4/mmm-CaAr3,P21/m-CaAr4andPm1-CaAr5were obtained. Our calculations show that the only stable phaseP63/mmc-CaAr can be synthesized at high pressure of 90 GPa. All the structures are ionic compounds of metallic nature, and surprisingly all Ar atoms attract electrons and act as an oxidant under high pressure conditions. The calculation results ofab initiomolecular dynamics show thatP63/mmc-CaAr compound maintains significant thermodynamic stability at high temperatures up to 1000 K. The high-pressure structures and electronic behaviors of the Ca-Ar system are expected to expand the understanding of the high-pressure chemical reactivity of compounds containing inert elements, and provide important theoretical support for the search of novel anomalous alkaline-earth metal inert element compounds.
RESUMO
Small GTPases including Ras, Rho, Rab, Arf, and Ran are omnipresent molecular switches in regulating key cellular functions. Their dysregulation is a therapeutic target for tumors, neurodegeneration, cardiomyopathies, and infection. However, small GTPases have been historically recognized as "undruggable". Targeting KRAS, one of the most frequently mutated oncogenes, has only come into reality in the last decade due to the development of breakthrough strategies such as fragment-based screening, covalent ligands, macromolecule inhibitors, and PROTACs. Two KRASG12C covalent inhibitors have obtained accelerated approval for treating KRASG12C mutant lung cancer, and allele-specific hotspot mutations on G12D/S/R have been demonstrated as viable targets. New methods of targeting KRAS are quickly evolving, including transcription, immunogenic neoepitopes, and combinatory targeting with immunotherapy. Nevertheless, the vast majority of small GTPases and hotspot mutations remain elusive, and clinical resistance to G12C inhibitors poses new challenges. In this article, we summarize diversified biological functions, shared structural properties, and complex regulatory mechanisms of small GTPases and their relationships with human diseases. Furthermore, we review the status of drug discovery for targeting small GTPases and the most recent strategic progress focused on targeting KRAS. The discovery of new regulatory mechanisms and development of targeting approaches will together promote drug discovery for small GTPases.
Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Descoberta de Drogas , ImunoterapiaRESUMO
Gold nanoclusters (AuNCs), as a novel fluorescent material, have been extensively explored and developed for bioimaging because of their attractive advantages such as ultrasmall size, low toxicity and exceptional two-photon excitation properties. However, it still remains a challenge to produce water-soluble, biocompatible and ultrabright AuNCs. Herein, we report on a novel one-pot synthesis of highly luminescent and biocompatible AuNCs by using polyvinyl pyrrolidone (PVP), a water-soluble polymer, to rigidify the primary stabilizing layer (shell) that is composed of 6-aza-2-thiothymine (ATT) ligands bound to the particle. Such shell-rigidification resulted in a significant enhancement of the fluorescence efficiency, reaching a quantum yield of 39% under the best conditions, about 35-fold increase from the intrinsically weak fluorescence of the AuNCs stabilized by only ATT. The fluorescence enhancement mechanism was systematically characterized, and the results indicate that PVP coating rigidifies the ATT ligand shell through steric hindrance and reduces the nonradiative relaxation of the excited states. The biocompatible PVP-AuNCs were further examined for two-photon cellular and sentinel lymph node (SLN) bioimaging, and we observed pH-dependent cytoplasmic images and intense green fluorescence in SLN and lymphatic vessels.
Assuntos
Ouro , Nanopartículas Metálicas , Ouro/química , Concentração de Íons de Hidrogênio , Luminescência , Nanopartículas Metálicas/química , ÁguaRESUMO
Immobilized G protein-coupled receptor is a versatile tool to study ligand-receptor interactions. In this work, we synthesized the immobilized alpha 1A adrenergic receptor (α1A-AR), a GPCR subtype mediating smooth muscle contraction, through a site-selective covalent method that relies on the reaction between haloalkane dehalogenase tagged α1A-AR and macroporous silica gel coated with 6-chlorohexanoic acid. To investigate thermodynamic and extra-thermodynamic parameters for ligand binding, we utilized the covalently immobilized receptor as stationary phase to perform frontal analysis and injection-amount dependent analysis as well as compared with the random immobilization method. Terazosin gave the association constant of 1.48 × 105 M-1 to α1A-AR, indicating that the oriented immobilization of α1A-AR enhances the ligand-binding activity by one order of magnitude in comparison with the random immobilization method (7.9 × 104 M-1). The binding of phentolamine and tamsulosin to the receptor was accompanied by a large absolute heat capacity (ΔCp) of 1.28 ± 0.23 kJ mol-1, demonstrating that the binding enthalpy and entropy appear to compensate for one another. These results indicated that the covalent immobilization of the receptor onto solid support has a profound impact on the ligand-binding activity of the receptor and the determination of ligand-receptor binding parameters. The receptor immobilized through the site-selective method will act as a benchmark for chromatographic determination of binding parameters in ligand-receptor interactions and can be used as an effective approach for rapid analysis of drug-protein interactions with high accuracy.
Assuntos
Cromatografia , Receptores Adrenérgicos alfa 1 , Ligantes , Tansulosina , TermodinâmicaRESUMO
The design, synthesis, and discovery of dual-target compounds are considered as a promising strategy to develop new drugs with improved safety and efficacy compared with single-target drugs. This necessitates development of the methodologies that enable us to rapidly and accurately achieve the dual-target leads. Applying rosmarinic acid, 18ß-glycyrrhetinic acid, rhein, and ferulic acid as template building blocks, we introduced the self-assembling DNA encoded technique to build the library containing 1,000 compounds. These compounds were screened by receptor chromatography with immobilized beta2-adrenoceptor (ß2-AR) and cysteinyl-leukotriene receptor (CysLT), whereby we obtained a derivative of 18ß-glycyrrhetinic acid (XC267) that specifically binds to the two receptors. In vitro assessment demonstrated the desired binding affinity of 6.57 × 104 M-1 to ß2-AR, 2.82 × 104 M-1 to CysLT, and the dissociation rate constant of 7.52 s-1 to ß2-AR, 17.2 s-1 to CysLT. Pharmacological examination with ovalbumin-induced mice demonstrated that XC267 significantly reduced the levels of IL-4, IL-13, and IgE after oral administration of 10 mg/kg. By Western blot analysis, we observed an up-regulated expression of ß2-AR and a blocked level of CysLT with a dose-dependent manner in pulmonary bronchial. Our results suggest XC627 is a potential candidate to treat asthma by simultaneously regulating the signaling pathway of the two receptors.
Assuntos
Asma , Produtos Biológicos , Animais , Asma/tratamento farmacológico , Cisteína , Leucotrienos , Ligantes , Camundongos , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/metabolismo , Transdução de SinaisRESUMO
Small GTPase or Ras superfamily, including Ras, Rho, Rab, Ran and Arf, are fundamental in regulating a wide range of cellular processes such as growth, differentiation, migration and apoptosis. They share structural and functional similarities for binding guanine nucleotides and hydrolyzing GTP. Dysregulations of Ras proteins are involved in the pathophysiology of multiple human diseases, however there is still a stringent need for effective treatments targeting these proteins. For decades, small GTPases were recognized as 'undruggable' targets due to their complex regulatory mechanisms and lack of deep pockets for ligand binding. NMR has been critical in deciphering the structural and dynamic properties of the switch regions that are underpinning molecular switch functions of small GTPases, which pave the way for developing new effective inhibitors. The recent progress of drug or lead molecule development made for small GTPases profoundly delineated how modern NMR techniques reshape the field of drug discovery. In this review, we will summarize the progress of structural and dynamic studies of small GTPases, the NMR techniques developed for structure-based drug screening and their applications in early-stage drug discovery for small GTPases.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Descoberta de Drogas , Humanos , Espectroscopia de Ressonância Magnética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas ras/metabolismoRESUMO
Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease (AD). There is considerable consensus that the increased production and/or aggregation of alpha-synuclein (alpha-syn) plays a central role in the pathogenesis of PD and related synucleinopathies. Current therapeutic strategies for treating PD offer mainly transient symptomatic relief and aim at the restitution of dopamine levels to counterbalance the loss of dopaminergic neurons. Therefore, the identification and development of drug-like molecules that block alpha-synuclein aggregation and prevent the loss of dopaminergic neurons are desperately needed to treat or slow the progression of PD. Here, we show that entacapone and tolcapone are potent inhibitors of alpha-syn and beta-amyloid (Abeta) oligomerization and fibrillogenesis, and they also protect against extracellular toxicity induced by the aggregation of both proteins. Comparison of the anti-aggregation properties of entacapone and tolcapone with the effect of five other catechol-containing compounds, dopamine, pyrogallol, gallic acid, caffeic acid, and quercetin on the oligomerization and fibrillization of alpha-syn and Abeta, demonstrate that the catechol moiety is essential for the anti-amyloidogenic activity. Our findings present the first characterization of the anti-amyloidogenic properties of tolcapone and entacapone against both alpha-synuclein and Abeta42 and highlight the potential of this class of nitro-catechol compounds as anti-amyloidogenic agents. Their inhibitory properties, mode of action, and structural properties suggest that they constitute promising lead compounds for further optimization.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Benzofenonas/farmacologia , Inibidores de Catecol O-Metiltransferase , Catecóis/farmacologia , Inibidores Enzimáticos/farmacologia , Nitrilas/farmacologia , Nitrofenóis/farmacologia , Fragmentos de Peptídeos/biossíntese , alfa-Sinucleína/biossíntese , Peptídeos beta-Amiloides/toxicidade , Animais , Microscopia Eletrônica , Ressonância Magnética Nuclear Biomolecular , Células PC12 , Fragmentos de Peptídeos/toxicidade , Ratos , TolcaponaRESUMO
Phosphorylation of alpha-synuclein (alpha-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating alpha-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate alpha-syn and beta-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate alpha-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate beta-syn at Ser-118, whereas no phosphorylation of gamma-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103-140) of alpha-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of alpha-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) alpha-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in alpha-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of alpha-syn and beta-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies.